ACS Publications. Most Trusted. Most Cited. Most Read
Quick View

OR SEARCH CITATIONS

My Activity
Publications
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Synth. Biol. 2021, 10, 10, 2552-2565
RETURN TO ISSUEPREVResearch ArticleNEXT

Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection

  • Elena Velázquez
    Elena Velázquez
    Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain
    More by Elena Velázquez
    Orcidhttps://orcid.org/0000-0001-6648-1634
  • , 
  • Yamal Al-Ramahi
    Yamal Al-Ramahi
    Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain
    More by Yamal Al-Ramahi
  • , 
  • Jonathan Tellechea-Luzardo
    Jonathan Tellechea-Luzardo
    Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.
    More by Jonathan Tellechea-Luzardo
  • , 
  • Natalio Krasnogor
    Natalio Krasnogor
    Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.
    More by Natalio Krasnogor
    Orcidhttps://orcid.org/0000-0002-2651-4320
  • , and 
  • Víctor de Lorenzo*
    Víctor de Lorenzo
    Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain
    *Email: [email protected]. Tel: 34-91 585 45 36. Fax: 34-91 585 45 06.
    More by Víctor de Lorenzo
    Orcidhttps://orcid.org/0000-0002-6041-2731
Cite this: ACS Synth. Biol. 2021, 10, 10, 2552–2565
Publication Date (Web):October 2, 2021
https://doi.org/10.1021/acssynbio.1c00199

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0.
  • Open Access

Article Views

2244

Altmetric

-

Citations

4
LEARN ABOUT THESE METRICS
PDF (5 MB)
Get e-Alerts
Supporting Info (1)»
SUBJECTS:

Abstract

High Resolution Image
Download MS PowerPoint Slide

Genome editing methods based on group II introns (known as targetron technology) have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination. Yet, their utility as delivery systems has typically been suboptimal due to the reduced efficiency of insertion when carrying exogenous sequences. We show that this limitation can be tackled and targetrons can be adapted as a general tool in Gram-negative bacteria. To this end, a set of broad-host-range standardized vectors were designed for the conditional expression of the Ll.LtrB intron. After establishing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths, to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. With these novel tools, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ΔrecA derivative. Finally, we applied this technology to successfully tag P. putida with an orthogonal short sequence barcode that acts as a unique identifier for tracking this microorganism in biotechnological settings. These results show the value of the targetron approach for the unrestricted delivery of small DNA fragments to precise locations in the genomes of Gram-negative bacteria, which will be useful for a suite of genome editing endeavors.

This publication is licensed under

CC-BY 4.0.
  • cc licence
  • by licence
KEYWORDS:
Pseudomonas putida is a soil bacterium and plant root colonizer that has emerged as one of the species with the highest potential as a synthetic biology chassis for industrial and environmental applications. (1,2) Qualities of interest include the lack of pathogenicity, (3) its high tolerance to oxidative stress (4,5) (a most desirable trait in processes such as biofuel production (6)), diverse and powerful capabilities for catabolizing aromatic compounds, (7−9) and ease of genetic and genomic manipulations. (10−13) In particular, a suite of molecular tools have become available for the deletion and insertion of exogenous sequences in the genome of this bacterium, directed either to random locations (e.g., with transposon vectors (14,15)) or to specific genomic loci through recombineering (16) or homologous recombination (13) (reviewed in ref (17)). In this last and most widely used case, recombination efficacies vary considerably among different bacterial groups and even strains of the same species. For example, editing in the archetypal P. putida KT2440 is particularly suboptimal in recA-dependent processes.
Group II introns could be an alternative editing technique in cases with low recombination-based editing efficiency, as they work in a recA-independent fashion. Group II introns are a type of retroelement with the capacity to self-splice from an mRNA and insert stably into specific DNA loci (a process known as retrohoming (18,19)). Their conserved tertiary structure and a protein encoded within the retroelement (IEP or intron-encoded protein) are key components for the splicing and recognition of the target DNA. (20,21) After translation, the IEP binds specifically to the catalytic RNA and first assists in the splicing of the intron from the containing exons. Afterward, both the IEP and the spliced intron stay together, forming a ribonucleoprotein (RNP) that carries out the recognition, reverse splicing, and retrotranscription of the intronic RNA into a new DNA site. (22) In the past, several of these introns have been engineered to recognize and insert into specific genes different from their native retrohoming sites, giving rise to the knockout system named targetron. (23) This platform is founded on the Ll.LtrB group II intron from Lactococcus lactis since it is the most studied case and it was proven to work in a wide range of bacterial genera, from Clostridium (24) or Bacillus (25) to the well-characterized species Escherichia coli. (23) Later, targetrons were assayed for the delivery of specific cargo sequences into designated loci. (26−28) Group II introns are promising tools to this end as they give rise to stable integrations. (26,28) Another useful feature is that they are broad-host-range actors that can work in a great variety of organisms. (24,25,27,29,30) Moreover, they can be redirected to virtually any desired genomic location with high specificity. (23,31) Finally, as already mentioned, they can be an alternative to homologous recombination-based techniques, as they function independently of recA, which is a considerable advantage compared to other editing systems. (32,33)
Unfortunately, the targetron system also has some shortcomings. First, targetrons can be modified to recognize new sequences, but integration efficiency greatly changes depending on the new target site. Algorithms have been developed to identify the best retargeting options in a given sequence for Ll.LtrB (23,24) and also for other group II introns. (31) These algorithms retrieve a list of integration loci near a target site, ordered by a predicted score, and they also design primers for the modification of the recognition sequences inside the intron. However, as a result of their probabilistic nature, these predictions are not always reliable. Second, while cargo sequences can be inserted inside of group II introns, e.g., in the IVb domain, (27) their addition lowers the efficiency of splicing and retrohoming. Therefore, despite the good characteristics of the platform, the application of group II introns as genome editing tools has not been widespread. Recently, some efforts to overcome these drawbacks were made when CRISPR/Cas9 technology was merged with targetrons to increase the recovery efficiency of successful mutants in E. coli. (33) In this case, successful targetron editing events are selected by exploiting the CRISPR/Cas9 machinery (34) as a way of counterselecting against nonmutated, wild-type sequences. (35,36) By designing gRNAs that recognize the target WT DNA and cleaving the cognate unedited genome site with Cas9/gRNA, the chance of finding correctly edited mutants increases greatly. However, the value of merging Ll.LtrB insertions with Cas9/gRNA counterselection and the tolerance of the system to exogenous cargos of different sizes have not been tackled thus far in other species. The design of a broad-host-range platform including all of the components of the system is thus required.
The work below describes a set of standardized plasmids expressing the Ll.LtrB intron under the control of different promoters (with IPTG or cyclohexanone induction (37)), origins of replication, and antibiotic resistance genes that all work in a suite of Gram-negative bacteria. To characterize the performance of the new expression plasmids, we engineered them to insert Ll.LtrB-carrying cargos of increasing sizes into specific genomic locations of E. coli and P. putida, in a fashion that can be easily counterselected for with the gRNA/Cas9 system mentioned above. (36) Furthermore, we show that the performance of the platform is independent of recA. Finally, we adopted this technology to successfully label P. putida KT2440 with a specific synthetic barcode for the identification and tracking of this strain in biotechnological applications. By introducing these barcodes, a physical link is created between the engineered organism and a digital twin. (39) As explained below, this enables a version control system for microbial strains where all important information can be archived and consulted whenever necessary.

Results and Discussion

ARTICLE SECTIONS
Jump To

Engineering Broad-Host Expression of Intron Ll.LtrB

The Ll.LtrB intron and commercial targetron technology (Supplementary Figure S1A) have been exploited to work in diverse bacteria, (23,24,27,29,30,38) but the vectors involved had to be modified in each case. In an attempt to make the same methodology more accessible and generalizable, we first set out to merge the key parts and properties of the Ll.LtrB system with standardized SEVA (Standard European Vector Architecture) (12,39,40) plasmids.
SEVA vectors are composed of interchangeable modules including broad-host-range origins of replication, antibiotic resistance genes, and a wide set of expression systems and reporter genes. To bring the SEVA standard to targetron technology, we constructed a collection of plasmids expressing the Ll.LtrB intron under the control of alternative expression systems and selectable markers that can be adapted for multiple purposes.
Figure 1 shows the whole experimental pipeline for the site-specific insertion of cargo in target genomic sites enabled by the pSEVA-Glli vector series. To benchmark the workflow, plasmid pSEVA421-GIIi (Km) was assembled (Supplementary Figure S1B). This construct is a low copy number (RK2 origin of replication) streptomycin/spectinomycin resistance plasmid carrying adjacent polycistronic Ll.LtrB intron and LtrA (Ll.LtrB IEP) sequences under the control of a T7 promoter. The Ll.LtrB segment of pSEVA421-GIIi (Km) has a retrotransposition-activation selectable marker (RAM) cargo composed of kanamycin resistance (KmR) gene interrupted by a group I intron (black square inside the KmR gene in the Supplementary Figure S1A,B,H) in domain IVb, which has been previously shown to increase the likelihood of finding retrohomed mutants. (41) The construct is arranged in a way that only if the Ll.LtrB intron is spliced and inserted, the group I intron is excised and the KmR gene is reconstituted. Therefore, selection in Km plates facilitates the identification of insertion mutants. Finally, the Ll.LtrB borne by pSEVA421-GIIi (Km) is retargeted to insert into position 1063 of the lacZ gene of E. coli in antisense orientation. The expectation is that upon correct Ll.LtrB retrohoming and insertion into a target genomic sequence (e.g., lacZ), the group I intron will be lost, the KmR restored, and the β-galactosidase gene interrupted, allowing for easy screening of the site-specific cargo insertion.

Figure 1

Figure 1. Diagram of targetrons and CRISPR/Cas9-mediated counterselection of insertions. The figure shows the technology developed in this article. Plasmid pSEVA-GIIi expresses the Ll.LtrB group II intron (empty or with cargo sequences cloned in the MluI site) and its IEP (LtrA) in the same transcriptional unit from the upstream promoter. The transcriptional unit is led by a retargeting region at 5′ (including exon 1 and the 5′ sequence of the Ll.LtrB intron), where three short sequences retrieved from the target gene (IBS, EBS1d, and EBS2) were engineered at given sites of the predicted transcript to secure its proper folding and retargeting (location of diagnostic primers is indicated). After transcription, the intronic RNA folds into a very conserved secondary structure and associates with LtrA to perform the splicing process from the exons. A lariat RNA is generated that remains attached to LtrA, forming a ribonucleoprotein (RNP) complex. This RNP scans DNA molecules until it finds the target site for retrohoming. Reverse splicing links covalently the intronic RNA to the sense strand of the DNA molecule, and the endonuclease (En) domain present in LtrA cleaves the antisense strand. Afterward, the retrotranscriptase (RT) domain of LtrA reverse transcribes the intronic RNA into DNA. The complete integration and synthesis of the cDNA is driven by repair mechanisms without the involvement of recombination. The incorporation of Cas9 complexes with gRNAs that recognize the insertion locus of Ll.LtrB causes the elimination of nonedited cells, as only those which incorporated the group II intron at the correct locus can survive (counterselection). IBS1: intron-binding site 1; EBS1d: exon-binding site 1-δ; EBS2: exon-binding site 2.

High Resolution Image
Download MS PowerPoint Slide
With this expectation, pSEVA421-GIIi (Km) was tested in E. coli BL21DE3 (Figure 2A–C). This strain was chosen as it provides the T7 RNAP (42) that is necessary for the expression of the whole plasmid insert. As the cloned group II intron was retargeted to insert into the lacZ gene, blue/white screening in X-gal plates was used to visually quantify the efficiency of the insertion process. A sample of the blue/white screenings of such insertion experiments with pSEVA421-GIIi (Km) is shown in Figure 2. The screens indicated that the Ll.LtrB intron was retrohoming to the selected locus inside the lacZ gene with good efficiency (Figure 2A). When Km was added to plates (Figure 2A, right plates), the number of white colonies was boosted in comparison to the plates with no selection (Figure 2A, left plates). Note that the induction of the T7 RNAP in the host strain E. coli BL21DE3 with IPTG did not make much difference in the frequencies of the process, which is likely due to the leakiness of the lacUV5 promoter that drives the expression of the polymerase. (43,44) In any case, the efficiency of the insertion of Ll.LtrB from pSEVA421-GIIi (Km) was calculated to be ca. 2.1 × 10–4 (in the +IPTG conditions).

Figure 2

Figure 2. SEVA plasmids encoding the Ll.LtrB group II intron and T7 RNAP work in E. coli BL21DE3 and P. putida KT2440. (A) Delivery of the Ll.LtrB intron from plasmid pSEVA421-GIIi (Km) in E. coli BL21DE3. The Ll.LtrB intron was retargeted to insert in the antisense orientation into the locus 1063 of the lacZ gene so that insertions would disrupt this gene, giving rise to white colonies in the presence of X-gal. Since a RAM is placed inside Ll.LtrB, kanamycin resistance was used as a way to select for intron insertion mutants (plates to the right). (B) Graph shows the number of KmR CFU normalized to 109 viable cells and classified according to the displayed phenotype in the presence of X-gal (blue colonies: lacZ+, blue bars; white colonies: lacZ– (disrupted), white hatched bars) and, also, according to the presence or absence of IPTG induction. (C) Representative colony PCR reactions to determine the correct insertion of Ll.LtrB inside the lacZ gene. Only if Ll.LtrB retrohomes, a PCR amplicon of 720 bp is generated. Blue numbers correspond to blue colonies, and black numbers correspond to white colonies used as the template material for each reaction. (D–F) Delivery of Ll.LtrB from plasmid pSEVA421-GIIi-pyrF and with the help of pSEVA131-T7RNAP in P. putida KT2440. (D) Bar plot showing the frequency of 5FOAR CFU normalized to 109 viable cells after the insertion assay. CFU are classified according to the addition or absence of IPTG during the incubation period. (E) Genuine efficiency of the insertion of Ll.LtrB. The proportion of uracil auxotrophs detected from the 5FOAR population was used as a ratio to determine the abundance of Ll.LtrB insertions in the population. (F) 5FOA counterselection was used to isolate insertion mutants that were not able to grow without uracil supplemented to plates. Colonies resistant to 5FOA but that were able to grow without uracil were used as negative controls of insertion. Two different PCR reactions are shown: (top gel) one primer annealed inside Ll.LtrB and the second annealed in the pyrF gene so that an amplicon could be only generated after intron insertion. (Bottom gel) two primers flanking the insertion locus were used so that two amplicons could be generated. The smallest fragment (380 bp) corresponds to the WT sequence, and the biggest fragment (∼1500 bp) corresponds to the insertion. The same colonies were tested in both PCR reactions. All bar graphs show the mean values (bars), standard deviation (lines), and single values (dots) of two biological replicates. WT: wild-type; Mut: insertion mutant; + control: reaction with a colony with successful insertion from a previous experiment used as a template; H2O: control PCR with no template material; UraR: colonies growing in media without uracil (no auxotrophs); UraS: colonies not growing in media without uracil (auxotrophs).

High Resolution Image
Download MS PowerPoint Slide
We also spotted a low number of KmR LacZ+ colonies (∼1% of total KmR clones) that was indicative of illegitimate off-target insertions, perhaps due to retrotransposition instead of retrohoming. (45−47) However, the frequency of such events was >200-fold less than those with the correct insertion phenotype (Figure 2B). Finally, colony PCR of white and blue colonies was performed to check the precise insertion of the group II intron into the lacZ gene and the correlation with the observed phenotype (Figure 2C). To this end, a total of 10 white colonies of the Km-containing plates were screened from each condition (induction versus noninduction), and all of them had incorporated Ll.LtrB in the expected site. Three KmR LacZ+ colonies from the same plates were also used as negative controls; all three failed to amplify, indicating no insertion of Ll.LtrB inside the lacZ gene.

Ll.LtrB Intron Retrohomes in P. putida KT2440

After testing the efficiency of the SEVA-based constructs described above in E. coli, we next inspected the activity of the same Ll.LtrB in P. putida. For this, we first constructed a compatible pSEVA vector for the heterologous expression of the T7 RNAP. The complete sequence of this ORF, along with the regulatory regions for IPTG-controlled expression (lacUV5 promoter along with a short 5′ region of the lacZ gene fused to the T7 RNAP ORF), was cloned into pSEVA131, yielding pSEVA131-T7RNAP (Supplementary Figure S1C). This plasmid bears an ampicillin resistance gene (ApR), a pBBR1 origin of replication, which confers a medium copy number of plasmids, and is compatible with pSEVA421-GIIi (Km). The target gene of choice for benchmarking the method in P. putida was not lacZ (which is absent in this species) but pyrF (PP1815), the orthologue of the yeast URA3 gene for orotidine-5′-phosphate decarboxylase (ODCase). (48) The loss of this marker can be selected both negatively (pyrF mutants become auxotrophic for uracil) and positively (the same mutants are also resistant to 5-fluoroorotic acid, 5FOA (49)). Since such a double selection works well in P. putida KT2440, (50) we adopted pyrF as a suitable candidate for the insertion of Ll.LtrB. For this, the Ll.LtrB was retargeted to insert into a specific locus inside the pyrF ORF, giving rise to pSEVA421-GIIi (Km)-pyrF. This plasmid is equivalent to the pSEVA421-GIIi (Km) used in E. coli (see above), but the retargeting region (Figure 1) was engineered to carry pyrF sequences instead of lacZ segments. A variant of this plasmid, which lacked the RAM used in the case of E. coli (see above), named pSEVA421-GIIi-pyrF (Supplementary Figure S1D), was also built to compare the efficacies of the different selection methods.
Both plasmids (i.e., with and without RAM) were transformed into P. putida KT2440 individually, in each case with pSEVA131-T7RNAP, and the insertion assay was performed as in E. coli above. After induction for 2 h with IPTG, the cultures were plated on a minimal medium supplemented with 5FOA and uracil to select for Ll.LtrB-retrohomed clones. While the frequency of 5FOAR spontaneous mutants in P. putida KT2440 (50) is ∼0.8 × 10–6, the intron insertion procedure increased the ratio of 5FOAR colonies to ∼10–4 (100-fold; Figure 2D). As in E. coli, IPTG addition increased only marginally the number of 5FOAR mutants. A number of individual 5FOAR clones were subsequently patched on plates with and without uracil to verify the pyrF-minus phenotype. Typically, 93.6% of 5FOAR clones from the noninduced culture and 86.2% of that with IPTG turned out to be uracil auxotrophs, which allowed the calculation of bona fide frequencies of Ll.LtrB insertion (Figure 2E). In addition, PCR reactions (Figure 2F) authenticated the accuracy of Ll.LtrB incorporation at the expected site in pyrF.
These experiments certified the ability of Ll.LtrB to retrohome into a specific site of the genome of P. putida KT2440 in a fashion similar to other Gram-negative species. (38) Intriguingly, selection of intron insertions based on RAM, which worked so well in E. coli (41) (see above) and other species, (51) failed altogether to deliver valid clones in P. putida, regardless of whether the selection was made with Km or 5FOA. Such a malfunction (which has been reported before in P. aeruginosa (38)) cannot be blamed on the lack of processivity of RNAP (the RNA element is expressed from a T7 promoter (42,52,53)). It is instead possible that the excision of the group I intron in the RAM may depend on host-specific factors.

Streamlining Ll.LtrB Expression and Activity in a ΔrecA Derivative Strain of P. putida KT2440

While the data above demonstrate the functioning of the Ll.LtrB platform in P. putida, the two-vector system can be simplified to make the platform easier to use in practice. To simplify the system to a single plasmid, we took advantage of the standardized architecture of SEVA plasmids to transfer the DNA segment of pSEVA421-GIIi-pyrF bearing Ll.LtrB and LtrA into pSEVA2311. (37) The result was the KmR and pBBR1 oriV plasmid called pSEVA2311-GIIi-pyrF (Supplementary Figure S1E), in which the expression of Ll.LtrB is under the control of the broad-host-range cyclohexanone-inducible ChnR/PChnB promoter device. (37,54) With this simplified tool in hand, we set out to compare the efficacy of Ll.LtrB insertion in the wild-type P. putida strain versus a recombination-deficient counterpart. To this end, P. putida KT2440 and an isogenic recA derivative were transformed with pSEVA2311-GIIi-pyrF, and the cultures of each transformant were grown and induced with 0, 0.5, 1, and 5 mM cyclohexanone. The samples were then plated on a minimal media with 5FOA and uracil to select for Ll.LtrB-inserted clones, and the colonies were analyzed as previously described. The results are shown in Figure 3. As in E. coli induced with IPTG, we found little difference in the number of 5FOAR CFUs induced without or with varying cyclohexanone concentrations (Figure 3A,C, left plots). In fact, no or low induction levels gave rise to the highest frequency of 5FOAR mutants in the ΔrecA mutant (Figure 3C, left plot). As previously described, 5FOAR colonies were patched on plates with and without uracil to search for authentic pyrF mutants, and the final frequency of 5FOAR/uracil auxotroph clones was calculated as an indication of the efficiency of insertion of Ll.LtrB for each cyclohexanone concentration (Figure 3A,C; right plots).

Figure 3

Figure 3. Performance of pSEVA2311-GIIi-pyrF in P. putida KT2440 and its ΔrecA derivative strain. (A) pSEVA2311-GIIi-pyrF works in P. putida KT2440 to deliver the Ll.LtrB intron into the pyrF gene through 5FOA counterselection. Left panel: Bar plot showing the frequency of 5FOAR CFUs normalized to 109 viable cells of wild-type P. putida after the insertion assay resulting from induction with various cyclohexanone concentrations (0, 0.5, 1, and 5 mM). Right panel: Genuine efficiency of insertion of Ll.LtrB. The proportion of uracil auxotrophs detected from the 5FOAR population was used as a reference to determine the abundance of Ll.LtrB insertions in each population. (B) Colony PCR reaction that used primers flanking the insertion locus was used to determine Ll.LtrB retrohoming in each concentration of cyclohexanone tested (from 0 mM in the left part of gel to 5 mM in the right part of gel). The smallest fragment (380 bp) corresponds to the wild-type sequence, while the biggest one (1500 bp) corresponds to the intron insertion in the correct location. (C, D) Functioning of pSEVA2311-GIIi-pyrF in P. putida KT2440 ΔrecA. The same experiments and analyses were done to determine the frequencies and correctness of intron insertion in the recombination-deficient strain. The gel at the bottom of (D) shows a control PCR reaction to verify recA minus genotype of the tested cells. The frequency of 5FOAR CFUs and the efficiency of insertion of Ll.LtrB in P. putida ΔrecA were determined as before. All bar graphs show the mean values (bars), standard deviation (lines), and single values (dots) of at least three biological replicates. WT, wild-type; Mut, insertion mutant; KT2440, parental strain; + control: PCR of DNA from an intron-inserted colony (from a previous experiment) used as the template; H2O: control PCR with no template material.

High Resolution Image
Download MS PowerPoint Slide
Finally, correct acquisition of the group II intron by pyrF was verified by means of colony PCR with primers flanking the site of insertion. Surprisingly, analysis of 5FOAR/uracil auxotroph colonies from both wild-type and ΔrecA cultures (Figure 3B,D) indicated that cyclohexanone induction did not help to generate more valid insertions. We speculate that excessive induction of the engineered intron RNA along with the LtrA complex encoded in plasmid can be toxic and/or produce unexpected effects. In any case, identification of Ll.LtrB insertions in a recA defective strain validated the ability of group II introns to work in a recombinant-independent fashion in P. putida. (32,55)

Merging Ll.LtrB Action with a CRISPR/Cas9-Mediated Counterselection System

Although the results above demonstrate the performance of group II intron insertion in P. putida, the measured frequencies of insertion were much too low to use the system as a practical genome editing tool when the pursued change is not selectable. On this basis, we sought to increase the insertion efficiency by combining the action of Ll.LtrB with the counterselection of unedited wild-type sequences with CRISPR/Cas9 (33,36)—all formatted with tools following the SEVA standard. The existing broad-host-range system of reference for such a counterselection involves compatible plasmids pSEVA231-CRISPR (KmR, pBBR1 oriV) for cloning the spacer and pSEVA421-Cas9tr (SmR, RK2 oriV) for the expression of Cas9 (Supplementary Table S2). This required the transfer of the DNA encoding the [ChnR/PChnB → Ll.LtrB/LtrA] device to a plasmid compatible with the other two. This process and the verification of the activity of the resulting intron delivery vectors are described in detail in the Supporting Information. The result of the exercise was pSEVA6511-GIIi-pyrF, as shown in Supplementary Figure S1F. This is a GmR plasmid with an RSF1010 oriV (12,56−58) compatible with pSEVA231-CRISPR and pSEVA421-Cas9tr and bearing a pyrF-retargeted Ll.LtrB and LtrA controlled by the ChnR/PChnB promoter.
With this 3-plasmid platform in hand, we first set out to explore the limits in the size of fragments that can be delivered by the Ll.LtrB intron in P. putida KT2440 and its ΔrecA derivative. Earlier work with other bacteria (26−28,33) indicated that the length of the sequences inserted inside Ll.LtrB was critical for the splicing and retrohoming efficiency of the intron. In fact, in its native host, L. lactis, cargo sequences >1 kb abolished retrohoming. (27) To determine the largest DNA fragment that Ll.LtrB was able to insert in both wild-type and ΔrecAP. putida, we generated a library of pSEVA6511-GIIi-pyrF plasmid derivatives carrying fragments of increasing size out of the luxC gene (from 150 to 1050 bp; Figure 4A). The maximum insertion size without CRISPR counterselection was first assessed by performing the assay on plates with and without 5FOA-mediated counterselection, with insert sizes of 150 bp (Lux1), 600 bp (Lux4), 750 bp (Lux5), and 1050 bp (Lux7). In the case of wild-type P. putida KT2440, 5FOA counterselection delivered correct insertions up to Lux4 (600 bp) but not larger segments (Supplementary Figure S2 and Supplementary Table S3). In contrast, only the smallest DNA cargo (150 bp) could be counterselected with 5FOA in P. putida ΔrecA (Supplementary Table S3).

Figure 4

Figure 4. Assessing the size-restriction of intron-mediated delivery with CRISPR/Cas9 counterselection using luxC fragments as a cargo. (A) Schematic of the intron library generated with increasing fragment length as a cargo (from 150 up to 1050 bp) using as template the first gene of the luxCDABEG operon, luxC. The Ll.LtrB intron in pSEVA6511-GIIi (LuxN) is retargeted to insert between the nucleotides 165 and 166 of the pyrF ORF in the antisense orientation. Spacer pyrF1 recognizes the region after the insertion site (part of the recognition site is shown inside a red box). The complementary nucleotides to the PAM (5′GGG-3′) are highlighted in red and are disrupted upon intron insertion. (B) Ll.LtrB-mediated delivery of luxC fragments in P. putida KT2440 WT with CRISPR/Cas9 counterselection. Colony PCR reactions showing amplifications from colonies with Ll.LtrB::LuxØ and Ll.LtrB::Lux1 (top gel) and corresponding PCR reactions verifying the recA-plus phenotype (bottom gel). WT amplification for the recA gene is 2 kb long. (C) Ll.LtrB-mediated delivery of luxC fragments in P. putida KT2440 ΔrecA. Colony PCR reactions showing amplifications from colonies with Ll.LtrB::LuxØ to Ll.LtrB::Lux4 (top gel) and corresponding PCR reaction verifying the recA—genotype (bottom gel). Deletion of the recA gene gives an amplification of 1 kb. WT: wild-type, Mut: insertion mutant, LuxN: cargos including from LuxØ to Lux7, Ø: Ll.LtrB with no cargo, 1: Ll.LtrB with Lux1 as the cargo, 2: Ll.LtrB with Lux2 as the cargo; 3: Ll.LtrB with Lux3 as the cargo, and 4: Ll.LtrB with Lux4 as the cargo. P. putida KT2440 ΔrecA colonies with no inserted Ll.LtrB used as the negative control.

High Resolution Image
Download MS PowerPoint Slide
We next measured the efficacy as a function of insert size of selecting the same Ll.LtrB intron insertions by means of CRISPR/Cas9-based counterselection, which is expected to kill the wild-type population of nonmodified cells. (33,35,36,59) For this, cells harboring both pSEVA421-Cas9tr (36) and the corresponding pSEVA6511-GIIi (LuxN)-pyrF were grown overnight and then induced for 4 h with cyclohexanone. After this incubation time, an aliquot of these cells was plated in the presence of 5FOA to estimate the efficiency of Ll.LtrB insertions with this protocol and no CRISPR/Cas9 counterselection, as these cells contained no plasmid carrying a guide RNA. The rest of the cells were made competent and then separately transformed with either pSEVA231-CRISPR (negative control with no specific spacer) pSEVA231-C-pyrF1 (bearing a specific spacer whose PAM occurs in the wild-type insertion locus of Ll.LtrB::LuxN in pyrF (36)). The cells were then plated on LB media supplemented with Sm and Km to select for clones with both pSEVA421-Cas9tr and pSEVA231-CRISPR or pSEVA231-C-pyrF1. Our prediction was that if Ll.LtrB::LuxN retrohomes were expected, the genomic PAM sequence within pyrF necessary for Cas9 activation would be disrupted, and only these mutated cells would be able to survive (Figure 4A). By following this approach, mutated cells in both P. putida wild-type (Figures 4B and 5A,C) and ΔrecA were isolated (Figures 4C and 5B,C). Interestingly, the wild-type strain did worse than the ΔrecA variant in accepting longer DNA segments as intron cargos. Under the best conditions, as shown in Figure 4C, the upper limit in the insert size for the ΔrecA strain was 600 bp (Lux4). As predicted, the frequency of correct insertions in either strain was boosted by pSEVA231-C-pyrF1 as compared to cells with the control plasmid pSEVA231-CRISPR (Supplementary Figure S3 and Supplementary Tables S4 and S5). In addition, counterselection allowed for the detection of longer inserts. In the absence of counterselection, no insertions of Ll.LtrB::Lux2–4 in the ΔrecA strain could be identified (Figure 5B and Supplementary Table S5). Furthermore, Ll.LtrB::Lux1 retrohoming in the wild-type strain could be detected only when pSEVA231-C-pyrF1 had been transformed with the other CRISPR counterselection plasmids (Figure 5A,C).

Figure 5

Figure 5. Intron insertion frequencies with CRISPR-Cas9 counterselection in wild-type and ΔrecAP. putida KT2440 confirmed through PCR. (A) Intron insertion frequency of each cargo inserted in the genome of WT P. putida KT2440 by Ll.LtrB using 5FOA; no counterselection (control plasmid pSEVA231-CRISPR) or CRISPR/Cas9-mediated counterselection (pSEVA231-C-pyrF1). Insertion of a cargo larger than Lux1 was not detected. (B) Similarly, for P. putida KT2440 ΔrecA, insertion of a cargo larger than Lux4 was not detected. (C) Combined efficiency of targetrons and CRISPR/Cas9 counterselection. The numbers of SmR KmR CFU obtained after transforming either pSEVA231-CRISPR (control) or pSEVA231-C-pyrF1 were normalized to 109 cells. The pSEVA231-CRISPR condition was set to 100%, and the percentage of cells with pSEVA231-C-pyrF1 was calculated accordingly. Finally, the ratio of positive Ll.LtrB insertions detected in the pSEVA231-C-pyrF1 condition was multiplied individually in each replicate. The mean (bars), single values (dots), and standard deviation (lines) of two or three replicates are shown. Ø: Ll.LtrB with no cargo; 1: Ll.LtrB with Lux1 as the cargo; 2: Ll.LtrB with Lux2 as the cargo; 3: Ll.LtrB with Lux3 as the cargo; and 4: Ll.LtrB with Lux4 as the cargo.

High Resolution Image
Download MS PowerPoint Slide
To estimate the efficiency of the merged targetrons/CRISPR/Cas9 system, the numbers of SmR KmR CFUs obtained after transforming either pSEVA231-CRISPR (control) or pSEVA231-C-pyrF1 to each strain were normalized to 109 viable cells (SmR CFUs). This control condition was set to 100% and the percentage of pSEVA231-C-pyrF1 was calculated accordingly. Finally, the ratio of positive Ll.LtrB insertions detected through PCR for each case was calculated and multiplied accordingly in each replicate (Figure 5C). Taken together, the results indicated that the system, within the cargo size limits, enables the positive selection of Ll.LtrB integrations with their cargo by killing the population of nonmutated bacteria. The higher frequency of insertions detected in the ΔrecA background is plausibly caused by the improved performance of CRISPR/Cas9 counterselection in recombination-deficient strains. (60)RecA-minus bacteria can neither use homologous recombination for repairing the double-stranded break catalyzed by Cas9 nor lose the CRISPR spacer cloned in pSEVA231-C-pyrF1, which is one of the main causes for escapers from the CRISPR/Cas9 counterselection to arise. (36) It should be noted that the insertion frequencies may change depending on the genomic target of the intron (33) and the sequence of the spacer in the CRISPR sequence.
Although the size of possible cargos that could be inserted in the P. putida genome was somewhat modest and the insertion frequencies decreased with their length, the results were in the range of those found in other bacterial types. (27,28) This highlights the value of the Ll.LtrB platform for delivery of small fragments to specific genomic loci in P. putida; a real-world example use case is addressed in the next section.

Application of Ll.LtrB for Barcoding Cells with Unique DNA Identifiers

A large number of biotechnological applications of P. putida could benefit from a targeted, stable genomic insertion of short synthetic and orthogonal DNA sequences (i.e., genetic barcodes) as unique identifiers of particular strains. These barcodes create a physical link between the tagged organism and a digital twin, (39) which in turn enables a version control system for microbial strains where all important information can be archived and consulted. (61,62) Given the broad host range and recombination-independent performance of the Ll.LtrB intron (27) for directed insertion of small fragments of DNA, we evaluated its value for delivery of such barcodes/unique identifiers to the genomes of strains of interest. The barcodes of choice have a small size (148 bp, Supplementary Figure S4) and they are composed of a universal primer (25 nt), which is shared by all barcodes generated with CellRepo software, (61) and a core sequence (123 nt). This is itself subdivided into three components: the barcode proper (96 nt), the synchronization sequence (9 nt), and the checksum (18 nt) component. (61)
The last two elements are incorporated as an error-correction mechanism. (61) In this way, even if truncated or incorrect reads are retrieved, the CellRepo algorithm is still able to identify the barcode and its linked strain profile content.
To exploit the Ll.LtrB/LtrA-based vector platform described above for targeting short sequences to the P. putida genome, a specific barcode generated with the CellRepo algorithm (61) was created with synthetic oligonucleotides (Supplementary Figure S4) and then cloned as a cargo of LI.LtrB in pSEVA6511-GIIi to generate pSEVA6511-GIIi (B3). Next, we searched for adequate targeting loci in the genome of P. putida. As barcodes are meant to link a strain to its digital data, they need to be included in a stable and permissive genetic locus, (61,62) such as intergenic regions close to essential genes. The context close to glmS (close to the att Tn7 site, Figure 6) was thus selected as a good candidate for the insertion of Ll.LtrB::B3. The Clostron algorithm (24) (http://www.clostron.com/) was used to survey the intergenic region between PP5408 and glmS to identify optimal targets. Two loci were picked from the retrieved list that were compatible with CRISPR/Cas9 counterselection (Figure 6 and Supplementary Figure S5), i.e., they contained a nearby PAM sequence in their vicinity in the correct orientation.

Figure 6

Figure 6. Application of the Ll.LtrB group II intron for delivery of specific genetic barcodes to the genome of P. putida KT2440. Organization of pSEVA6511-GIIi (B3) variants is shown with the intron retargeted toward Locus 1 (x = 37s) or Locus 2 (x = 95a). Selection of the insertion loci for Ll.LtrB::B3 is in the vicinity of the Tn7-insertion site (black triangle). Two different insertion points (gray triangles) were chosen for the insertion list generated on the Clostron website, (24) and Ll.LtrB::B3 was retargeted to both sites accordingly. The recognition site in Locus 1 (green) is located in the sense strand, while Locus 2 (orange) is present in the antisense strand of the P. putida genome. Ll.LtrB::B3 insertion would generate two different genotypes depending on the locus being targeted in each case.

High Resolution Image
Download MS PowerPoint Slide
Then, the retargeting region of Ll.LtrB was separately engineered to these two loci, giving rise to pSEVA6511-GIIi(B3)-37s and pSEVA6511-GIIi(B3)-94a, respectively (Figure 6). In parallel, specific CRISPR spacers for either locus were designed and tested to measure the efficiency of CRISPR/Cas9 cleavage, which was >90% in both cases (Supplementary Figure S6). After these two components were ready, the same insertion protocol we used before for luxC fragments was adopted for the delivery of the barcodes. Thereby, the obtained colonies were directly checked through pool PCR reactions for correct barcode insertion, as no phenotype change was expected after the insertion of the cargo-containing Ll.LtrB. Additional PCRs were performed to secure the purity of isolated colonies (data not shown), and barcode integrity was confirmed through sequencing. Only in the case of Locus 2, bona fide insertions were found to bear the corresponding barcode sequence (Figure 7). Details about the final barcoded strain can be found in the public CellRepo repository: https://cellrepo.ico2s.org/repositories/93?branch_id=139&locale=en.

Figure 7

Figure 7. Delivery of Ll.LtrB::B3 containing a barcode in the P. putida KT2440 genome. A first pool PCR was set to detect successful Ll.LtrB::B3 insertions in either Locus 1 (green) or Locus 2 (orange). The top gel shows the amplification found with a pool PCR using primers flanking the insertion at Locus 2. The bottom gel shows the second PCR of individual colonies from the corresponding pool to find the barcoded clone. In this case, a primer annealing inside the barcode (pbarcode universal) and another annealing inside the PP5408 gene were used. WT: wild-type, Locus 1: 36,37s insertion site, Locus 2: 94,95a insertion site. PP5408 (green gene), glmS (orange gene).

High Resolution Image
Download MS PowerPoint Slide

Conclusions

ARTICLE SECTIONS
Jump To
While P. putida KT2440 has made evident its utility in biotechnological applications, there is still a need to develop new tools that can be used to modify this bacterium and broaden its applicability. Moreover, testing these broad-host-range tools in P. putida showcases their potential for modifying a suite of other Gram-negative bacteria of interest, especially those deficient in homologous recombination. This work is an attempt to expand the number of genetic assets that can be exploited to insert sequences of certain lengths at specific genomic regions, in this case using group II introns. Given the functionality of these retroelements through virtually all of the evolutionary trees (22,63) and the orthogonality of their DNA insertion mechanism, (24,30,38) we argue that they are ideal devices for the incorporation of standardized short sequences in a wide variety of biological destinations. One specific application is the insertion of unique identifiers for barcoding strains (39) and other live items of biotechnological interest, for the sake of traceability and securing intellectual property. (64) While the SEVA-based system described here is ready to use in Pseudomonas and other Gram-negative bacteria, the minimal delivery device formed by the Ll.LtrB element and the LtrA protein complex can be easily moved to any other platform optimized for other recipients. We argue that this work contributes to the developing collection of trans-kingdom genetic tools for the insertion of DNA sequences in a range of biological systems while using the same delivery mechanism.

Methods

ARTICLE SECTIONS
Jump To

Bacterial Strains and Media

E. coli CC118 strain [Δ(ara-leu), araD139, ΔlacX74, galE, galK phoA20, thi-1, rpsE, rpoB, argE (Am), recA1, OmpC+, OmpF+] was used for plasmid cloning and propagation and BL21DE3 strain [fhuA2, [lon], ompT, gal, (λ DE3), [dcm], ΔhsdS; (λ DE3) = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5] for intron mobility assays in E. coli. P. putida KT2440 and its derivative ΔrecA were used to assess intron mobility in this species. Luria-Bertani (LB) medium was used for general growth and was supplemented when needed with kanamycin (Km; 50 μg/mL), ampicillin (Ap; 150 μg/mL for E. coli and 500 μg/mL for P. putida), gentamycin (Gm; 10 μg/mL for E. coli and 15 μg/mL for P. putida), and/or streptomycin (Sm; 50 μg/mL for E. coli and 100 μg/mL for P. putida). For solid plates, LB medium was supplemented with 1.5% agar (w/v). In specific cases for P. putida, M9 minimal medium [6 g/L Na2HPO4, 3 g/L KH2PO4, 1.4 g/L (NH4)2SO4, 0.5 g/L NaCl, 0.2 g/L MgSO4·7H2O] supplemented with sodium citrate at 0.2% (w/v) as the carbon source was used instead. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) was added at a final concentration of 30 μg/mL to carry out blue/white colony screening. Moreover, different inducers were added to media when necessary: isopropyl-1-thio-b-galactopyranoside (IPTG) and cyclohexanone at 1 mM unless stated otherwise.

Plasmid Construction

The complete sequence encoding the T7 promoter, Ll.LtrB intron, and LtrA protein was amplified from the commercial plasmid pACD4K-C (TargeTron gene knockout system, Sigma-Aldrich) with primers pGIIintron_fwd and rev (Supplementary Table S1). The amplified fragment was then digested with PacI and SpeI restriction enzymes and cloned into a similarly digested pSEVA427, yielding pSEVA421-GIIi (Km). The lacUV5 promoter along with T7 RNA polymerase (T7 RNAP) sequences was amplified from pAR1219 (Merck) with primers pAR1219_fwd and rev, PacI/SpeI digested, and cloned into corresponding sites of pSEVA131, generating pSEVA131-T7RNAP, necessary for the transcription of the Ll.LtrB intron from the T7 promoter. To eliminate the retrotransposition-activated selectable marker (RAM) present inside Ll.LtrB, pSEVA421-GIIi (Km) was digested with the MluI restriction enzyme and then directly ligated and transformed to obtain pSEVA421-GIIi. To change the expression system and simplify the intron expression mechanism, only Ll.LtrB (with or without RAM) and LtrA sequences were extracted by HindIII/SpeI digestion of pSEVA421-GIIi and cloned into pSEVA2311, giving rise to pSEVA2311-GIIi (Km) and pSEVA2311-GIIi, respectively. These plasmids have both the Ll.LtrB intron and LtrA expression controlled under the ChnR-PChnB promoter. Finally, to assemble an expression plasmid compatible with the CRISPR/Cas9 system described previously, (36) it was necessary to modify both the origin of replication and the antibiotic resistance gene. For that, the ChnR-PChnB promoter, Ll.LtrB (with or without RAM), and LtrA sequences were extracted by digestion with PacI/SpeI enzymes and cloned into pSEVA651 equivalent sites to obtain pSEVA6511-GIIi (Km) and pSEVA6511-GIIi. The CRISPR/Cas9 counterselection approach used in this work was described in ref (36) and was based on plasmids pSEVA421-Cas9tr and pSEVA231-CRISPR. pSEVA231-C-pyrF1 was generated and described in the same work. The rest of the spacers for counterselection were designed manually and cloned into BsaI-digested pSEVA231-CRISPR, following the protocol explained in the same paper. The resulting plasmids were named pSEVA231-C-37s and pSEVA231-C-94a.

Retargeting of the Ll.LtrB Intron

Retargeting of the Ll.LtrB intron was performed by adapting the Targetron protocol from Sigma-Aldrich. First, the Clostron platform (24) (http://www.clostron.com/) was used to design primers pIBS-X, pEBS1d-X, pEBS2-X, and pEBSuniversal (depending on the insertion target; Supplementary Table S1) with corresponding target sequences as query (lacZ gene in E. coli; pyrF gene and PP5408-glmS region in P. putida). From the output list, the best-ranked targets compatible with CRISPR/Cas9 technology were selected in each case. This means targets with PAM sequences (5′-NGG-3′ in the case of the Streptococcus pyogenes system) closest to the insertion site of the intron were chosen. Afterward, Clostron-designed oligonucleotides for each target were used in a SOEing PCR (65) with pACD4K-C as a template to yield a 350 bp fragment. For the cloning of this amplicon, different strategies were adopted according to the final recipient plasmid. For the retargeting of pSEVA421-GIIi and its derivatives, the fragment was digested with BsrGI/HindIII restriction enzymes and ligated into the linearized recipient plasmid. For retargeting of pSEVA2311-GIIi and pSEVA6511 derivatives, Gibson assembly (66,67) was chosen as the cloning procedure since an additional BsrGI restriction site was present in the pChnB promoter. Primers pRetarget-fwd and rev were used to reamplify the SOEing amplicon and add the corresponding homologous sequences to directly assemble the fragment to HindIII/HpaI-digested pSEVA2311/6511-GIIi.

Insertion of Exogenous Sequences Inside Ll.LtrB

All exogenous sequences inserted inside the Ll.LtrB intron were cloned into the MluI site present in the intron sequence. In the case of the insert to be delivered, two strategies were followed: the luxC gene from the lux operon was employed as a template for the generation of fragments of different sizes (from 150 to 1050 bp with a difference of 150 bp each). Primer pLux_fwd in combination with primer pLux1–7_rev (Supplementary Table S1) was used in a PCR step to generate each fragment using as template pSEVA256. Each amplicon was then digested with MluI and cloned into linearized pSEVA6511-GIIi-pyrF. The sense orientation of each fragment was confirmed by sequencing. Barcode sequences were created on the CellRepo website (https://cellrepo.herokuapp.com) with an algorithm that provides universally unique identifiers (UUIDs). (61) This provides the possibility to produce a large library of barcodes that are randomly generated and unique. After selecting one specific barcode, a BLAST search was done to make sure there was no other region with high similarity in the genome of P. putida. Once a barcode was verified, it was generated by a PCR step with 119-mer oligonucleotides bearing 30 overlapping nucleotides at 3′. These primers also included 30 nucleotides complementary to the recipient vector at 5′, so a Gibson assembly reaction (66,67) could be performed after amplification with MluI-digested pSEVA6511-GIIi.

Interference Assay of Spacers 37s and 94a

P. putida KT2440 strain harboring pSEVA421-Cas9tr was grown overnight, and electrocompetent cells were prepared by washing cells with 300 mM sucrose a total of five times. The final pellet was resuspended in 400 μL and then split into 100 μL aliquots. One hundred nanograms of pSEVA231-CRISPR (control), pSEVA231-C-37s, or pSEVA231-C-94a (Supplementary Table S2) was electroporated into respective aliquots. Transformed bacteria were grown in LB/Sm for 2 h at 30 °C and serial dilutions were then plated on LB/Sm to test viability and on LB/Sm/Km plates to assess the efficiency of cleavage. After counting CFUs under both conditions, the ratio of transformation efficiency was calculated by dividing the CFUs on LB/Sm/Km plates by CFUs on LB/Sm plates and normalized to 109 cells.

Ll.LtrB Insertion Assay in E. coli

Briefly, cells harboring the corresponding pSEVA-GIIi derivative plasmid were grown in LB supplemented with the corresponding antibiotics. When an OD of 0.2 was reached, the right inducer was added to the medium and the cells were incubated at 30 °C for different periods (from 30 min to 4 h depending on the expression system). When IPTG was used, the cells were washed and recuperated in fresh media after the induction period of 1 h at 30 °C. Finally, serial dilutions were plated to assess viability and intron insertion efficiency on selective media as indicated in each case.

Ll.LtrB Insertion Assay in P. putida

The same protocol described above for E. coli was used with P. putida strains, with the only difference that the induction time was 2 or 4 h (depending on the expression system), and no recovery was performed after 4 h induction. Also, as pyrF gene was the target of Ll.LtrB insertion, the cells were plated on M9 minimal media supplemented with only 20 μg/mL uracil (Ura) to assess viability on uracil and 250 μg/mL 5-fluoroorotic acid (5FOA) to counterselect pyrF-disruption mutants and make easier the identification of insertion events. An average of 50 colonies per condition were patched on minimal media with and without uracil to assess the proportion of uracil auxotrophs among the 5FOAR colonies.

CRISPR/Cas9 Counterselection Assay in P. putida KT2440 and KT2440 ΔrecA

When CRISPR/Cas9 counterselection was to be applied, the protocol was modified to simplify the process. The cells harboring both pSEVA421-Cas9tr and pSEVA6511-GIIi derivatives were grown overnight at 30 °C. The next day, 1 mM cyclohexanone was added to the culture, and the cells were induced for 4 h at 30 °C. After this incubation, 1 mL of cells was plated on M9 minimal media supplemented with Ura and 5FOA to assess the native efficiency of insertion in this condition. Later, the cells were made electrocompetent, and 100 ng of pSEVA231-CRISPR or pSEVA231-C-spacer (pyrF1, 37s or 94a depending on the experiment) was electroporated. Finally, the cells were recovered in LB/Sm for 2 h at 30 °C, after which serial dilutions were plated on LB/Sm (to assess viability) and LB/Sm/Km (to assess counterselection efficiency).

Calculation of Merged Targetrons/CRISPR/Cas9 Efficiency

For the generation of this ratio, the normalized frequency of SmR KmR CFU obtained per 109 viable cells (SmR) after transforming pSEVA231-CRISPR or pSEVA231-C-pyrF1 was first calculated with the formula
Next, the ratio of counterselection efficiency (R) was calculated by dividing the normalized SmR KmR CFU obtained with pSEVA231-C-pyrF1 by the one calculated for pSEVA231-CRISPR.
Finally, the efficiency of the merged Ll.LtrB insertion and CRISPR/Cas9 counterselection was obtained after multiplying the percentage of positive clones verified through PCR in the pSEVA231-C-pyrF1 condition. The final ratio was plotted as a percentage
These formulae were used with each cargo size and in each replicate individually. The error and standard deviation were calculated based on the efficiency of targetron + CRISPR system ratio of each replicate using GraphPad Prism 6 (https://www.graphpad.com).

Analysis of Ll.LtrB Insertion by Colony PCR

Ll.LtrB integrations were studied by colony PCR to check the presence or absence of the intron in the correct loci. Two possible reactions were used: in one, primers flanking the insertion site to amplify the whole group II intron were used. The product of this PCR would be composed of the intron sequence and the amplified flanking regions. In the second, one primer was annealed in the target locus and the other inside the intron sequence; consequently, a PCR product was only obtained when the Ll.LtrB intron was present. In the case of barcode delivery, pool PCR reactions with a total of four colonies per reaction were first performed, followed by PCR of individual colonies in positive pools. PCRs were analyzed by electrophoresis on agarose gel and 1× TAE (Tris-Acetate-EDTA). EZ Load 500 bp Molecular Ruler (Brio-Rad) was the DNA ladder in all gels.

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.1c00199.

  • List of oligonucleotides used in this study (Supplementary Table S1); list of plasmids used in this work (Supplementary Table S2); insertion frequencies of Ll.LtrB::Lux1 and Ll.LtrB::Lux4 in P. putida KT2440 WT and ΔrecA with no CRISPR/Cas9-mediated counterselection (Supplementary Table S3); insertion frequency of Ll.LtrB::LuxN intron in P. putida KT2440 WT with 5FOA CRISPR/Cas9-mediated counterselection (Supplementary Table S4); insertion frequency of Ll.LtrB::LuxN intron in P. putida KT2440 ΔrecA with 5FOA CRISPR/Cas9-mediated counterselection (Supplementary Table S5); construction and verification of intron delivery plasmids compatible with CRISPR/Cas9-mediated counterselection (Supplementary Methods); pSEVA plasmids for the expression of the Ll.LtrB intron in a wide range of Gram-negative bacteria (Supplementary Figure S1); preliminary approach to assess size-restriction of intron-mediated delivery using luxC fragments as a cargo and 5FOA counterselection in log-phase induced cells (Supplementary Figure S2); total intron insertion frequency without differentiating the cargo that was being delivered (Supplementary Figure S3); barcode generation with a PCR using 3′-overlapping 119-mer oligonucleotides (Supplementary Figure S4); application of targetrons as a barcode delivery system (Supplementary Figure S5); and design and test of Locus 1 and 2 spacers for CRISPR/Cas9-mediated counterselection of Ll.LtrB::B3 group II intron (Supplementary Figure S6) (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

ARTICLE SECTIONS
Jump To
  • Corresponding Author
  • Authors
    • Elena Velázquez - Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain;  Orcidhttps://orcid.org/0000-0001-6648-1634
    • Yamal Al-Ramahi - Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain
    • Jonathan Tellechea-Luzardo - Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.
    • Natalio Krasnogor - Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.;  Orcidhttps://orcid.org/0000-0002-2651-4320
  • Author Contributions

    E.V., V.d.L., Y.A.-R., and N.K. planned the experiments; E.V. and J.T.-L. did the practical work. All authors analyzed and discussed the data and contributed to the writing of the article.

  • Funding

    This work was funded by the SETH (RTI2018-095584-B-C42) (MINECO/FEDER), SyCoLiM (ERA-COBIOTECH 2018—PCI2019-111859-2) Project of the Spanish Ministry of Science and Innovation. MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018-820699), SynBio4Flav (H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650) and MIX-UP (MIX-UP H2020-BIO-CN-2019-870294) Contracts of the European Union and the InGEMICS-CM (S2017/BMD-3691) Project of the Comunidad de Madrid—European Structural and Investment Funds—(FSE, FECER). E.V. was the recipient of a Fellowship from the Education Ministry, Madrid, Spanish Government (FPU15/04315). N.K. acknowledges the Engineering and Physical Sciences Research Council (EPSRC) grant “Synthetic Portabolomics: Leading the way at the crossroads of the Digital and the Bio Economies (EP/N031962/1)”, and the Royal Academy of Engineering Chair in Emerging Technologies award.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

ARTICLE SECTIONS
Jump To

The authors are indebted to Prof. Chris Miller (UC Denver) for critical reading of the manuscript. Figure 1 includes an image retrieved from https://biorender.com/ and used under subscription 89B77366-0004.

References

ARTICLE SECTIONS
Jump To

This article references 67 other publications.

  1. 1
    Nikel, P. I.; de Lorenzo, V. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism. Metab. Eng. 2018, 50, 142– 155,  DOI: 10.1016/j.ymben.2018.05.005
    Google Scholar
    1
    Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism
    Nikel, Pablo I.; de Lorenzo, Victor
    Metabolic Engineering (2018), 50 (), 142-155CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
    The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochem. stresses. Over the years, these properties have been capitalized biotechnol. owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biol. have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biol. systems. Much of the biotechnol. interest on P. putida stems from the distinct architecture of its central carbon metab. The native biochem. is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochem. network of P. putida (cis-metab.) has sufficed to obtain target compds. of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metab.) for making complex, new-to-Nature mols. A no. of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufg. of fine and bulk chems. in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochem. beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chem. elements into the extant biochem., as well as genomic stability and scaling-up issues.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpvVSlt7k%253D&md5=fac6e8937d2e4f3d0c26bcde01e6777c
  2. 2
    Nikel, P. I.; Martínez-García, E.; de Lorenzo, V. Biotechnological domestication of pseudomonads using synthetic biology. Nat. Rev. Microbiol. 2014, 12, 368– 379,  DOI: 10.1038/nrmicro3253
    Google Scholar
    2
    Biotechnological domestication of pseudomonads using synthetic biology
    Nikel, Pablo I.; Martinez-Garcia, Esteban; de Lorenzo, Victor
    Nature Reviews Microbiology (2014), 12 (5), 368-379CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Much of contemporary synthetic biol. research relies on the use of bacterial chassis for plugging-in and plugging-out genetic circuits and new-to-nature functionalities. However, the microorganisms that are the easiest to manipulate in the lab. are often suboptimal for downstream industrial applications, which can involve physicochem. stress and harsh operating conditions. In this Review, we advocate the use of environmental Pseudomonas strains as model organisms that are pre-endowed with the metabolic, physiol. and stress-endurance traits that are demanded by current and future synthetic biol. and biotechnol. needs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlahtL8%253D&md5=d2cc342c2b17f5631572b664f5513acb
  3. 3
    Kampers, L. F. C.; Volkers, R. J. M.; Martins Dos Santos, V. A. P. Pseudomonas putida KT2440 is HV1 certified, not GRAS. Microb. Biotechnol. 2019, 12, 845– 848,  DOI: 10.1111/1751-7915.13443
    Google Scholar
    3
    Pseudomonas putida KT2440 is HV1 certified, not GRAS
    Kampers Linde F C; Volkers Rita J M; Martins Dos Santos Vitor A P; Martins Dos Santos Vitor A P
    Microbial biotechnology (2019), 12 (5), 845-848 ISSN:.
    Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M3msleqsA%253D%253D&md5=01a803206ea94a4286be8ab8a039549c
  4. 4
    Kim, J.; Park, W. Oxidative stress response in Pseudomonas putida. Appl. Microbiol. Biotechnol. 2014, 98, 6933– 6946,  DOI: 10.1007/s00253-014-5883-4
    Google Scholar
    4
    Oxidative stress response in Pseudomonas putida
    Kim, Jisun; Park, Woojun
    Applied Microbiology and Biotechnology (2014), 98 (16), 6933-6946CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    A review. Pseudomonas putida is widely distributed in nature and is capable of degrading various org. compds. due to its high metabolic versatility. The survival capacity of P. putida stems from its frequent exposure to various endogenous and exogenous oxidative stresses. Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents existing in various niches. ROS could facilitate the evolution of bacteria by mutating genomes. Aerobic bacteria maintain defense mechanisms against oxidative stress throughout their evolution. To overcome the detrimental effects of oxidative stress, P. putida has developed defensive cellular systems involving induction of stress-sensing proteins and detoxification enzymes as well as regulation of oxidative stress response networks. Genetic responses to oxidative stress in P. putida differ markedly from those obsd. in Escherichia coli and Salmonella spp. Two major redox-sensing transcriptional regulators, SoxR and OxyR, are present and functional in the genome of P. putida. However, the novel regulators FinR and HexR control many genes belonging to the E. coli SoxR regulon. Oxidative stress can be generated by exposure to antibiotics, and iron homeostasis in P. putida is crucial for bacterial cell survival during treatment with antibiotics. This review highlights and summarizes current knowledge of oxidative stress in P. putida, as a model soil bacterium, together with recent studies from mol. genetics perspectives.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVamtLbN&md5=de993728cab8409fdd59ed5c00fcb724
  5. 5
    Nikel, P. I.; Fuhrer, T.; Chavarría, M.; Sánchez-Pascuala, A.; Sauer, U.; de Lorenzo, V. Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress. ISME J. 2021, 15, 1751– 1766,  DOI: 10.1038/s41396-020-00884-9
    Google Scholar
    5
    Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress
    Nikel, Pablo I.; Fuhrer, Tobias; Chavarria, Max; Sanchez-Pascuala, Alberto; Sauer, Uwe; de Lorenzo, Victor
    ISME Journal (2021), 15 (6), 1751-1766CODEN: IJSOCF; ISSN:1751-7362. (Nature Portfolio)
    As a frequent inhabitant of sites polluted with toxic chems., the soil bacterium and plant-root colonizer Pseudomonas putida can tolerate high levels of endogenous and exogenous oxidative stress. Yet, the ultimate reason of such phenotypic property remains largely unknown. To shed light on this question, metabolic network-wide routes for NADPH generation-the metabolic currency that fuels redox-stress quenching mechanisms-were inspected when P. putida KT2440 was challenged with a sub-lethal H2O2 dose as a proxy of oxidative conditions. 13C-tracer expts., metabolomics, and flux anal., together with the assessment of physiol. parameters and measurement of enzymic activities, revealed a substantial flux reconfiguration in oxidative environments. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidn., and the cyclic operation of the pentose phosphate pathway led to significant NADPH-forming fluxes, exceeding biosynthetic demands by ∼50%. The resulting NADPH surplus, in turn, fueled the glutathione system for H2O2 redn. These properties not only account for the tolerance of P. putida to environmental insults-some of which end up in the formation of reactive oxygen species-but they also highlight the value of this bacterial host as a platform for environmental bioremediation and metabolic engineering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVSisLc%253D&md5=31d1780e11c16b4bbdbf09c008f4324d
  6. 6
    Liao, J. C.; Mi, L.; Pontrelli, S.; Luo, S. Fuelling the future: Microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 2016, 14, 288– 304,  DOI: 10.1038/nrmicro.2016.32
    Google Scholar
    6
    Fuelling the future: microbial engineering for the production of sustainable biofuels
    Liao, James C.; Mi, Luo; Pontrelli, Sammy; Luo, Shanshan
    Nature Reviews Microbiology (2016), 14 (5), 288-304CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one soln. is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the prodn. of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compds. and how to optimize biofuel prodn. by engineering fuel pathways and central metab.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XltVaqsbk%253D&md5=fcd67bab889558ff6f918a5d70a9d6ec
  7. 7
    Jiménez, J. I.; Miñambres, B.; García, J. L.; Díaz, E. Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environ. Microbiol. 2002, 4, 824– 841,  DOI: 10.1046/j.1462-2920.2002.00370.x
    Google Scholar
    7
    Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440
    Jimenez, Jose Ignacio; Minambres, Baltasar; Garcia, Jose Luis; Diaz, Eduardo
    Environmental Microbiology (2002), 4 (12), 824-841CODEN: ENMIFM; ISSN:1462-2912. (Blackwell Science Ltd.)
    Anal. of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural arom. compds. and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central arom. intermediates, i.e., the protocatechuate (pca genes) and catechol (cat genes) branches of the β-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two addnl. gene clusters that might be involved in the catabolism of N-heterocyclic arom. compds. (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compds. (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually assocd. with the gene clusters, a supraoperonic clustering of catabolic genes that channel different arom. compds. into a common central pathway (catabolic island) was not obsd. in P. putida KT2440. The global view on the mineralization of arom. compds. by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegrdn./biotransformation processes, and reveals this bacterium as a useful model system for studying biochem., genetic, evolutionary and ecol. aspects of the catabolism of arom. compds.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvVGrtg%253D%253D&md5=1922f12af80e022107400b58fa47f419
  8. 8
    Ramos, J. L.; Duque, E.; Huertas, M. J.; Haidour, A. Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons. J. Bacteriol. 1995, 177, 3911– 3916,  DOI: 10.1128/jb.177.14.3911-3916.1995
    Google Scholar
    8
    Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons
    Ramos, Juan L.; Duque, Estrella; Huertas, Maria-Jose; Haidour, Ali
    Journal of Bacteriology (1995), 177 (14), 3911-16CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to org. solvents whose log Pow (octanol/water partition coeff.) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg2+ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concns. of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarog. studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolize toluene. Although the strain also thrived in high concns. of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmvV2gsb0%253D&md5=92a84eea5b4f2a8abba19a91b534c80b
  9. 9
    Martínez, I.; Mohamed, M. E. S.; Rozas, D.; García, J. L.; Díaz, E. Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds. Metab. Eng. 2016, 35, 46– 54,  DOI: 10.1016/j.ymben.2016.01.005
    Google Scholar
    9
    Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds
    Martinez, Igor; Mohamed, Magdy El-Said; Rozas, Daniel; Garcia, Jose Luis; Diaz, Eduardo
    Metabolic Engineering (2016), 35 (), 46-54CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
    The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of arom. heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compd. dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) mol. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial "chassis" for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative - to the previously constructed recombinant dsz cassettes - for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and once secreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient prodn. of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlalu70%253D&md5=6021bcdce6cb854cabf99a30150e080a
  10. 10
    Martínez-García, E.; Nikel, P. I.; Aparicio, T.; de Lorenzo, V. Pseudomonas 2.0: Genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb. Cell Fact. 2014, 13, 159  DOI: 10.1186/s12934-014-0159-3
    Google Scholar
    10
    Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression
    Martinez-Garcia, Esteban; Nikel, Pablo I.; Aparicio, Tomas; de Lorenzo, Victor
    Microbial Cell Factories (2014), 13 (), 159/1-159/33CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
    Background: Because of its adaptability to sites polluted with toxic chems., the model soil bacterium Pseudomonas putida is naturally endowed with a no. of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a deriv. strain in which the genome has been heavily edited in order to construct a robust microbial cell factory. Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiol. vigour. Conclusion: The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches (e.g., increasing the promoter strength in the DNA constructs of interest).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1Oqt7g%253D&md5=dfebf084cef553ec5d6526f9fbbd504f
  11. 11
    Lieder, S.; Nikel, P. I.; de Lorenzo, V.; Takors, R. Genome reduction boosts heterologous gene expression in Pseudomonas putida. Microb. Cell Fact. 2015, 14, 23  DOI: 10.1186/s12934-015-0207-7
    Google Scholar
    11
    Genome reduction boosts heterologous gene expression in Pseudomonas putida
    Lieder Sarah; Takors Ralf; Nikel Pablo I; de Lorenzo Victor
    Microbial cell factories (2015), 14 (), 23 ISSN:.
    BACKGROUND: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome). RESULTS: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440. CONCLUSIONS: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MjlvVeqtA%253D%253D&md5=1b3829a02ebcccfb6bd3f5dfc0299255
  12. 12
    Silva-Rocha, R.; Martínez-García, E.; Calles, B.; Chavarría, M.; Arce-Rodríguez, A.; De Las Heras, A.; Páez-Espino, A. D.; Durante-Rodríguez, G.; Kim, J.; Nikel, P. I.; Platero, R.; De Lorenzo, V. The Standard European Vector Architecture (SEVA): A coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 2013, 41, D666– D675,  DOI: 10.1093/nar/gks1119
    Google Scholar
    12
    The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes
    Silva-Rocha, Rafael; Martinez-Garcia, Esteban; Calles, Belen; Chavarria, Max; Arce-Rodriguez, Alejandro; de las Heras, Aitor; Paez-Espino, A. David; Durante-Rodriguez, Gonzalo; Kim, Juhyun; Nikel, Pablo I.; Platero, Raul; de Lorenzo, Victor
    Nucleic Acids Research (2013), 41 (D1), D666-D675CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    The Std. European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical lab. strains. Under the SEVA std., every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with phys. connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvV2ktrzF&md5=3ddd60ae5099d577aa6b688813a5605b
  13. 13
    Martínez-García, E.; de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: Analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 2011, 13, 2702– 2716,  DOI: 10.1111/j.1462-2920.2011.02538.x
    Google Scholar
    13
    Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440
    Martinez-Garcia, Esteban; de Lorenzo, Victor
    Environmental Microbiology (2011), 13 (10), 2702-2716CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
    The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, the authors employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-neg. bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation expts. revealed that the procedure did not increase mutation rates - perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand the authors made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to det. various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Anal. of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-neg. microorganisms.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVKhtbfP&md5=8a2204c9c029721125df30a7c2ac80e2
  14. 14
    Martínez-García, E.; De Lorenzo, V. Transposon-based and plasmid-based genetic tools for editing genomes of Gram-negative bacteria. Methods Mol. Biol. 2012, 813, 267– 283
    Google Scholar
    14
    Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria
    Martinez-Garcia, Esteban; de Lorenzo, Victor
    Methods in Molecular Biology (New York, NY, United States) (2012), 813 (Synthetic Gene Networks), 267-283CODEN: MMBIED; ISSN:1064-3745. (Springer)
    A good part of the contemporary synthetic biol. agenda aims at reprogramming microorganisms to enhance existing functions and/or perform new tasks. Moreover, the functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavors rely on the availability of genetic tools to successfully modify a la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for the prodn. of directed mutants and allelic replacements, random mutant libraries to discover new functions, and means to stably implant larger genetic networks into the genome of specific hosts. The list of gram-neg. species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available mol. techniques to do so is very limited beyond Escherichia coli. In this chapter, utilization of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of gram-neg. microorganisms.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Sqs7jK&md5=d8dc7fef841f41065b1e0c236b3339d8
  15. 15
    Martínez-García, E.; Aparicio, T.; de Lorenzo, V.; Nikel, P. I. New transposon tools tailored for metabolic engineering of Gram-negative microbial cell factories. Front. Bioeng. Biotechnol. 2014, 2, 46  DOI: 10.3389/fbioe.2014.00046
    Google Scholar
    15
    New transposon tools tailored for metabolic engineering of gram-negative microbial cell factories
    Martinez-Garcia Esteban; Aparicio Tomas; de Lorenzo Victor; Nikel Pablo I
    Frontiers in bioengineering and biotechnology (2014), 2 (), 46 ISSN:2296-4185.
    Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. A la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3nsVOrtw%253D%253D&md5=e535341c401daaac2882e3d09ba452b3
  16. 16
    Aparicio, T.; Nyerges, A.; Martínez-García, E.; de Lorenzo, V. High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering. iScience 2020, 23, 100946  DOI: 10.1016/j.isci.2020.100946
    Google Scholar
    16
    High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering
    Aparicio, Tomas; Nyerges, Akos; Martinez-Garcia, Esteban; de Lorenzo, Victor
    iScience (2020), 23 (3), 100946CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
    Application of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnol. relevant strain EM42 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutLPPE36K was designed under the control of the PL/cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments-e.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering of P. putida, as required for metabolic reprogramming of this important synthetic biol. chassis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsl2mtLg%253D&md5=7b4bae6ce59f76035cfa133e40ac0be5
  17. 17
    Martin-Pascual, M.; Batianis, C.; Bruinsma, L.; Asin-Garcia, E.; Garcia-Morales, L.; Weusthuis, R. A.; van Kranenburg, R.; Martins Dos Santos, V. A. P. A navigation guide of synthetic biology tools for Pseudomonas putida. Biotechnol. Adv. 2021, 49, 107732  DOI: 10.1016/j.biotechadv.2021.107732
    Google Scholar
    17
    A navigation guide of synthetic biology tools for Pseudomonas putida
    Martin-Pascual, Maria; Batianis, Christos; Bruinsma, Lyon; Asin-Garcia, Enrique; Garcia-Morales, Luis; Weusthuis, Ruud A.; van Kranenburg, Richard; Martins dos Santos, Vitor A. P.
    Biotechnology Advances (2021), 49 (), 107732CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)
    Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnol., owing to its remarkable metabolic versatility and ability to sustain difficult redox reactions and operational stresses, among other attractive characteristics. A wealth of genetic and in silico tools have been developed to enable the unravelling of its physiol. and improvement of its performance. However, the rise of this microbe as a promising platform for biotechnol. applications has resulted in diversification of tools and methods rather than standardization and convergence. As a consequence, multiple tools for the same purpose have been generated, while most of them have not been embraced by the scientific community, which has led to compartmentalization and inefficient use of resources. Inspired by this and by the substantial increase in popularity of P. putida, we aim herein to bring together and assess all currently available (wet and dry) synthetic biol. tools specific for this microbe, focusing on the last 5 years. We provide information on the principles, functionality, advantages and limitations, with special focus on their use in metabolic engineering. Addnl., we compare the tool portfolio for P. putida with those for other bacterial chassis and discuss potential future directions for tool development. Therefore, this review is intended as a ref. guide for experts and new 'users' of this promising chassis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1yrt7bL&md5=3012891782213362ce45205f20b9ddfe
  18. 18
    Mörl, M.; Niemer, I.; Schmelzer, C. New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates. Cell 1992, 70, 803– 810,  DOI: 10.1016/0092-8674(92)90313-2
    Google Scholar
    18
    New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates
    Morl M; Niemer I; Schmelzer C
    Cell (1992), 70 (5), 803-10 ISSN:0092-8674.
    Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate. These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK38zos1Gquw%253D%253D&md5=0de88e6dcdb98d1f7c6c68cdac24d25e
  19. 19
    Lazowska, J.; Meunier, B.; Macadre, C. Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers. EMBO J. 1994, 13, 4963– 4972,  DOI: 10.1002/j.1460-2075.1994.tb06823.x
    Google Scholar
    19
    Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers
    Lazowska, Jaga; Meunier, Brigitte; Macadre, Catherine
    EMBO Journal (1994), 13 (20), 4963-72CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
    Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COX1 gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was obsd. to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behavior of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhvF2hsrY%253D&md5=c7313b7d97832fa83b5ed5c1ba63683b
  20. 20
    Saldanha, R.; Chen, B.; Wank, H.; Matsuura, M.; Edwards, J.; Lambowitz, A. M. RNA and protein catalysis in group II intron splicing and mobility reactions using purified components. Biochemistry 1999, 38, 9069– 9083,  DOI: 10.1021/bi982799l
    Google Scholar
    20
    RNA and Protein Catalysis in Group II Intron Splicing and Mobility Reactions Using Purified Components
    Saldanha, Roland; Chen, Bing; Wank, Herbert; Matsuura, Manabu; Edwards, Judy; Lambowitz, Alan M.
    Biochemistry (1999), 38 (28), 9069-9083CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles contg. only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed anal. of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a Kd of ≤0.12 pM at 30°. This binding occurs in a rapid bimol. reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochem. anal. of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVCqtro%253D&md5=2136004d61355a78d3ad72c72f646545
  21. 21
    Matsuura, M.; Noah, J. W.; Lambowitz, A. M. Mechanism of maturase-promoted group II intron splicing. EMBO J. 2001, 20, 7259– 7270,  DOI: 10.1093/emboj/20.24.7259
    Google Scholar
    21
    Mechanism of maturase-promoted group II intron splicing
    Matsuura, Manabu; Noah, James W.; Lambowitz, Alan M.
    EMBO Journal (2001), 20 (24), 7259-7270CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
    Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chem. modification expts. that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyneratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsF2h&md5=18a1b077ef7c4155b12d2fc0bef65540
  22. 22
    Lambowitz, A. M.; Zimmerly, S. Group II introns: Mobile ribozymes that invade DNA. Cold Spring Harbor Perspect. Biol. 2011, 3, a003616  DOI: 10.1101/cshperspect.a003616
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Perutka, J.; Wang, W.; Goerlitz, D.; Lambowitz, A. M. Use of Computer-designed Group II Introns to Disrupt Escherichia coli DExH/D-box Protein and DNA Helicase Genes. J. Mol. Biol. 2004, 336, 421– 439,  DOI: 10.1016/j.jmb.2003.12.009
    Google Scholar
    23
    Use of Computer-designed Group II Introns to Disrupt Escherichia coli DExH/D-box Protein and DNA Helicase Genes
    Perutka, Jiri; Wang, Wenjun; Goerlitz, David; Lambowitz, Alan M.
    Journal of Molecular Biology (2004), 336 (2), 421-439CODEN: JMOBAK; ISSN:0022-2836. (Elsevier)
    Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the assocd. intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small no. of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E. coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational anal. provides new insight into group II intron target site recognition, and the set of E. coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntVKrsw%253D%253D&md5=d7cde4aede8256f94d4df7eeed98ed2b
  24. 24
    Heap, J. T.; Pennington, O. J.; Cartman, S. T.; Carter, G. P.; Minton, N. P. The ClosTron: A universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods 2007, 70, 452– 464,  DOI: 10.1016/j.mimet.2007.05.021
    Google Scholar
    24
    The ClosTron: A universal gene knock-out system for the genus Clostridium
    Heap, John T.; Pennington, Oliver J.; Cartman, Stephen T.; Carter, Glen P.; Minton, Nigel P.
    Journal of Microbiological Methods (2007), 70 (3), 452-464CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)
    Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the no. of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpsFKjsLk%253D&md5=5ad07920edc8de3b8f622522223016aa
  25. 25
    Akhtar, P.; Khan, S. A. Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis. Plasmid 2012, 67, 111– 117,  DOI: 10.1016/j.plasmid.2011.12.012
    Google Scholar
    25
    Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis
    Akhtar, Parvez; Khan, Saleem A.
    Plasmid (2012), 67 (2), 111-117CODEN: PLSMDX; ISSN:0147-619X. (Elsevier)
    The large pXO1 plasmid (181.6 kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two sep. regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivs. contg. only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy no. anal. showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xks12jtrY%253D&md5=e29997ac16445fd79197fc6dbd55ccb6
  26. 26
    Frazier, C. L.; San Filippo, J.; Lambowitz, A. M.; Mills, D. A. Genetic manipulation of Lactococcus lactis by using targeted group II introns: Generation of stable insertions without selection. Appl. Environ. Microbiol. 2003, 69, 1121– 1128,  DOI: 10.1128/AEM.69.2.1121-1128.2003
    Google Scholar
    26
    Genetic manipulation of Lactococcus lactis by using targeted group II introns: Generation of stable insertions without selection
    Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.
    Applied and Environmental Microbiology (2003), 69 (2), 1121-1128CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
    Despite their com. importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to sep. deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtF2it7w%253D&md5=70f040e4515aded320ec667e68c53cce
  27. 27
    Plante, I.; Cousineau, B. Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron. RNA 2006, 12, 1980– 1992,  DOI: 10.1261/rna.193306
    Google Scholar
    27
    Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron
    Plante, Isabelle; Cousineau, Benoit
    RNA (2006), 12 (11), 1980-1992CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    The Ll.LtrB intron, from the low G+C gram-pos. bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addn. of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant redn. in lariat accumulation in L. lactis. This suggests that cargo-contg. Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-contg. Ll.LtrB variants that are mobile.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFygsL%252FF&md5=12308f25b4b51b486da6ce6398af0b73
  28. 28
    Rawsthorne, H.; Turner, K. N.; Mills, D. A. Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl. Environ. Microbiol. 2006, 72, 6088– 6093,  DOI: 10.1128/AEM.02992-05
    Google Scholar
    28
    Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences
    Rawsthorne, Helen; Turner, Kevin N.; Mills, David A.
    Applied and Environmental Microbiology (2006), 72 (9), 6088-6093CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
    Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here the authors describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, resp., were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the resp. tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy no. of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same no. of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was obsd. from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addn., the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVKjt70%253D&md5=c1d1e67be17b38c6d7a699b3b11ca654
  29. 29
    Yao, J.; Zhong, J.; Fang, Y.; Geisinger, E.; Novick, R. P.; Lambowitz, A. M. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA 2006, 12, 1271– 1281,  DOI: 10.1261/rna.68706
    Google Scholar
    29
    Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing
    Yao, Jun; Zhong, Jin; Fang, Yuan; Geisinger, Edward; Novick, Richard P.; Lambowitz, Alan M.
    RNA (2006), 12 (7), 1271-1281CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivs. of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a deriv. of pCN37 with a temp.-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the prodn. of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temp.-sensitive, this method yields a conditional hsa disruptant that grows at 32° but not 43°. The temp. sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temp. sensitivity of group II intron splicing could limit the temp. range of an organism contg. a group II intron inserted in an essential gene.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xms1Cqu78%253D&md5=4e089632c8b7390ccce87c3513cbe723
  30. 30
    Karberg, M.; Guo, H.; Zhong, J.; Coon, R.; Perutka, J.; Lambowitz, A. M. Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat. Biotechnol. 2001, 19, 1162– 1167,  DOI: 10.1038/nbt1201-1162
    Google Scholar
    30
    Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria
    Karberg, Michael; Guo, Huatao; Zhong, Jin; Coon, Robert; Perutka, Jiri; Lambowitz, Alan M.
    Nature Biotechnology (2001), 19 (12), 1162-1167CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here the authors show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptV2qsrw%253D&md5=3dad957336fc3ad3a37f4316d019bf4a
  31. 31
    García-Rodríguez, F. M.; Hernańdez-Gutiérrez, T.; Diáz-Prado, V.; Toro, N. Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting. RNA Biol. 2014, 11, 391– 401,  DOI: 10.4161/rna.28373
    Google Scholar
    31
    Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting
    Garcia-Rodriguez Fernando M; Hernandez-Gutierrez Teresa; Diaz-Prado Vanessa; Toro Nicolas
    RNA biology (2014), 11 (4), 391-401 ISSN:.
    Gene-targeting vectors derived from mobile group II introns capable of forming a ribonucleoprotein (RNP) complex containing excised intron lariat RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT), maturase, and endonuclease (En) activities have been described. RmInt1 is an efficient mobile group II intron with an IEP lacking the En domain. We performed a comprehensive study of the rules governing RmInt1 target site recognition based on selection experiments with donor and recipient plasmid libraries, with randomization of the elements of the intron RNA involved in target recognition and the wild-type target site. The data obtained were used to develop a computer algorithm for identifying potential RmInt1 targets in any DNA sequence. Using this algorithm, we modified RmInt1 for the efficient recognition of DNA target sites at different locations in the Sinorhizobium meliloti chromosome. The retargeted RmInt1 integrated efficiently into the chromosome, regardless of the location of the target gene. Our results suggest that RmInt1 could be efficiently adapted for gene targeting.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crlsFehsA%253D%253D&md5=49269dde7cb61dfd2adf91c2aa0d3971
  32. 32
    Cousineau, B.; Smith, D.; Lawrence-Cavanagh, S.; Mueller, J. E.; Yang, J.; Mills, D.; Manias, D.; Dunny, G.; Lambowitz, A. M.; Belfort, M. Retrohoming of a bacterial group II intron: Mobility via complete reverse splicing, independent of homologous DNA recombination. Cell 1998, 94, 451– 462,  DOI: 10.1016/S0092-8674(00)81586-X
    Google Scholar
    32
    Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination
    Cousineau, Benoit; Smith, Dorie; Lawrence-Cavanagh, Stacey; Mueller, John E.; Yang, Jian; Mills, David; Dunny, Gary; Lambowitz, Alan M.; Belfort, Marlene
    Cell (Cambridge, Massachusetts) (1998), 94 (4), 451-462CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The mobile group II intron of Lactococcus lactis, LI.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that LI.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homol. and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, LI.LtrB has many features of retrotransposons, with practical and evolutionary implications.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlslOktb8%253D&md5=ebbed8461cc1dadf9c870f01354bac6d
  33. 33
    Velázquez, E.; Lorenzo, V. D.; Al-Ramahi, Y. Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery. ACS Synth. Biol. 2019, 8, 2186– 2193,  DOI: 10.1021/acssynbio.9b00293
    Google Scholar
    33
    Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery
    Velazquez, Elena; Lorenzo, Victor de; Al-Ramahi, Yamal
    ACS Synthetic Biology (2019), 8 (9), 2186-2193CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Group II introns were developed time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-pos. and Gram-neg. bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of addnl. genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counter-selection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed proved that directing CRISPR/Cas9 towards the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counter selection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2ntbzN&md5=f06edd98e918993b500d50caa1978a9c
  34. 34
    Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007, 315, 1709– 1712,  DOI: 10.1126/science.1138140
    Google Scholar
    34
    CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
    Barrangou, Rodolphe; Fremaux, Christophe; Deveau, Helene; Richards, Melissa; Boyaval, Patrick; Moineau, Sylvain; Romero, Dennis A.; Horvath, Philippe
    Science (Washington, DC, United States) (2007), 315 (5819), 1709-1712CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addn. of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with assocd. cas genes, provided resistance against phages, and resistance specificity is detd. by spacer-phage sequence similarity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtlWntb8%253D&md5=b88c0100d2c0469213afda20e47c39cd
  35. 35
    Jiang, W.; Bikard, D.; Cox, D.; Zhang, F.; Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 2013, 31, 233– 239,  DOI: 10.1038/nbt.2508
    Google Scholar
    35
    RNA-guided editing of bacterial genomes using CRISPR-Cas systems
    Jiang, Wenyan; Bikard, David; Cox, David; Zhang, Feng; Marraffini, Luciano A.
    Nature Biotechnology (2013), 31 (3), 233-239CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-assocd. Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFCkurY%253D&md5=ea3bbfda133b51e71b114de2c9ceabfd
  36. 36
    Aparicio, T.; de Lorenzo, V.; Martínez-García, E. CRISPR/Cas9-Based Counterselection Boosts Recombineering Efficiency in Pseudomonas putida. Biotechnol. J. 2018, 13, 1700161  DOI: 10.1002/biot.201700161
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Benedetti, I.; Nikel, P. I.; de Lorenzo, V. Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria. Data Brief 2016, 6, 738– 744,  DOI: 10.1016/j.dib.2016.01.022
    Google Scholar
    37
    Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria
    Benedetti Ilaria; Nikel Pablo I; de Lorenzo Victor
    Data in brief (2016), 6 (), 738-44 ISSN:2352-3409.
    Engineering of robust microbial cell factories requires the use of dedicated genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We have edited and formatted the ChnR/P chnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The proposed compositional standard was thoroughly verified with a monomeric and superfolder green fluorescent protein (msf•GFP) in Escherichia coli. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Data in this article are related to the research paper "Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes" [1].
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jgt1Onug%253D%253D&md5=5c2853ffbcc263263df26ad415e9484a
  38. 38
    Yao, J.; Lambowitz, A. M. Gene targeting in Gram-negative bacteria by use of a mobile group II intron (“targetron”) expressed from a broad-host-range vector. Appl. Environ. Microbiol. 2007, 73, 2735– 2743,  DOI: 10.1128/AEM.02829-06
    Google Scholar
    38
    Gene targeting in gram-negative bacteria by use of a mobile group II intron ("targetron") expressed from a broad-host-range vector
    Yao, Jun; Lambowitz, Alan M.
    Applied and Environmental Microbiology (2007), 73 (8), 2735-2743CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
    Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector contg. an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-neg. bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvFeqsbc%253D&md5=136a1d18e1873775be03c8940174d9fe
  39. 39
    Martínez-García, E.; Aparicio, T.; Goñi-Moreno, A.; Fraile, S.; De Lorenzo, V. SEVA 2.0: An update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic Acids Res. 2015, 43, D1183– D1189,  DOI: 10.1093/nar/gku1114
    Google Scholar
    39
    SEVA 2.0: an update of the standard European vector architecture for de-/re-construction of bacterial functionalities
    Martinez-Garcia, Esteban; Aparicio, Tomas; Goni-Moreno, Angel; Fraile, Sofia; de Lorenzo, Victor
    Nucleic Acids Research (2015), 43 (D1), D1183-D1189CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    A review. The Std. European Vector Architecture 2.0 database (SEVA-DB 2.0) is an improved and expanded version of the platform released in 2013 aimed at assisting the choice of optimal genetic tools for deconstructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA std. facilitates combinations of functional DNA segments that ease both the anal. and the engineering of diverse Gram-neg. bacteria for fundamental or biotechnol. purposes. The large no. of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biol. Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biol. communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVymtLfL&md5=8152ca1366af4c0fe1d99435b137bbcb
  40. 40
    Martínez-García, E.; Goñi-Moreno, A.; Bartley, B.; McLaughlin, J.; Sánchez-Sampedro, L.; Pascual Del Pozo, H.; Prieto Hernández, C.; Marletta, A. S.; De Lucrezia, D.; Sánchez-Fernández, G.; Fraile, S.; De Lorenzo, V. SEVA 3.0: An update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts. Nucleic Acids Res. 2020, 48, D1164– D1170,  DOI: 10.1093/nar/gkz1024
    Google Scholar
    40
    SEVA 3.0: an update of the standard European vector architecture for enabling portability of genetic constructs among diverse bacterial hosts
    Martinez-Garcia, Esteban; Goni-Moreno, Angel; Bartley, Bryan; McLaughlin, James; Sanchez-Sampedro, Lucas; del Pozo, Hector Pascual; Hernandez, Clara Prieto; Marletta, Ada Serena; De Lucrezia, Davide; Sanchez-Fernandez, Guzman; Fraile, Sofia; de Lorenzo, Victor
    Nucleic Acids Research (2020), 48 (D1), D1164-D1170CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A review. The Std. European Vector Architecture 3.0 database is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for anal., construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several tech., computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biol. Open Language) format. This ensures interoperability with other platforms and affords simulations of their behavior under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biol. approaches towards non-std. bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnol. endeavours.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1GltrbP&md5=4f5a11f9bf0a49d09a3ff86bcfdd1252
  41. 41
    Zhong, J.; Karberg, M.; Lambowitz, A. M. Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res. 2003, 31, 1656– 1664,  DOI: 10.1093/nar/gkg248
    Google Scholar
    41
    Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker
    Zhong, Jin; Karberg, Michael; Lambowitz, Alan M.
    Nucleic Acids Research (2003), 31 (6), 1656-1664CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron contg. a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Anal. of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy no. of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXit1yks7g%253D&md5=9ddea609f3357b85ce578dbb1967c657
  42. 42
    Studier, F. W.; Moffatt, B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 1986, 189, 113– 130,  DOI: 10.1016/0022-2836(86)90385-2
    Google Scholar
    42
    Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
    Studier, F. William; Moffatt, Barbara A.
    Journal of Molecular Biology (1986), 189 (1), 113-30CODEN: JMOBAK; ISSN:0022-2836.
    A gene expression system based on phage T7 RNA polymerase [9014-24-8] was developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amt. of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for rRNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and(or) stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degrdn. Apparently, a specific mRNA produced by T7 RNA polymerase can rapidly sat. the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to >50% of the total cell protein in ≤3 h. Two ways were used to deliver active T7 RNA polymerase to the cell: (1) infection by a λ deriv. that carried gene 1; or (2) induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silently in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable. But if the target plasmid can be maintained, induction of chromosomal gene 1 can be a convenient way to produce large amts. of target RNA and(or) protein. T7 RNA polymerase seems to be capable of transcribing almost any DNA linked to a T7 promoter, so the T7 expression system should be capable of transcribing almost any gene or its complement in E. coli. Comparable T7 expression systems can be developed in other types of cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XktlKrsr4%253D&md5=3219300bc2f640fe9830c7518eb99bfc
  43. 43
    Dubendorf, J. W.; Studier, F. W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 1991, 219, 45– 59,  DOI: 10.1016/0022-2836(91)90856-2
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Angius, F.; Ilioaia, O.; Amrani, A.; Suisse, A.; Rosset, L.; Legrand, A.; Abou-Hamdan, A.; Uzan, M.; Zito, F.; Miroux, B. A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins. Sci. Rep. 2018, 8, 8572  DOI: 10.1038/s41598-018-26668-y
    Google Scholar
    44
    A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins
    Angius Federica; Ilioaia Oana; Amrani Amira; Suisse Annabelle; Rosset Lindsay; Legrand Amelie; Abou-Hamdan Abbas; Uzan Marc; Zito Francesca; Miroux Bruno; Suisse Annabelle; Abou-Hamdan Abbas
    Scientific reports (2018), 8 (1), 8572 ISSN:.
    Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mbkt12isg%253D%253D&md5=0256960f3095dbbacbc5be9d233b3c38
  45. 45
    Ichiyanagi, K.; Beauregard, A.; Lawrence, S.; Smith, D.; Cousineau, B.; Belfort, M. Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets. Mol. Microbiol. 2002, 46, 1259– 1272,  DOI: 10.1046/j.1365-2958.2002.03226.x
    Google Scholar
    45
    Retrotransposition of the LI.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets
    Ichiyanagi, Kenji; Beauregard, Arthur; Lawrence, Stacey; Smith, Dorie; Cousineau, Benoit; Belfort, Marlene
    Molecular Microbiology (2002), 46 (5), 1259-1272CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
    Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double-stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, LI.LtrB, supported frequent use of RNA targets during retrotransposition, recent expts. with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single-stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron-encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging-strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides crit. for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging-strand template, hinting at a role for DNA replication in intron dispersal in nature.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsFGisL0%253D&md5=a588926c206e0bf19be2da6bb66a36c9
  46. 46
    Dickson, L.; Huang, H. R.; Liu, L.; Matsuura, M.; Lambowitz, A. M.; Perlman, P. S. Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13207– 13212,  DOI: 10.1073/pnas.231494498
    Google Scholar
    46
    Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites
    Dickson, Lorna; Huang, Hon-Ren; Liu, Lu; Matsuura, Manabu; Lambowitz, Alan M.; Perlman, Philip S.
    Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (23), 13207-13212CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addn. to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXosFygtrs%253D&md5=5bd9e382b0d94d8b6bb91f15232e59cc
  47. 47
    Coros, C. J.; Landthaler, M.; Piazza, C. L.; Beauregard, A.; Esposito, D.; Perutka, J.; Lambowitz, A. M.; Belfort, M. Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment. Mol. Microbiol. 2005, 56, 509– 524,  DOI: 10.1111/j.1365-2958.2005.04554.x
    Google Scholar
    47
    Retrotransposition strategies of the lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment
    Coros, Colin J.; Landthaler, Markus; Piazza, Carol Lyn; Beauregard, Arthur; Esposito, Donna; Perutka, Jiri; Lambowitz, Alan M.; Belfort, Marlene
    Molecular Microbiology (2005), 56 (2), 509-524CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
    Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Addnl., growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjsFWmtLs%253D&md5=d68f89a37bc9cd4bd4c9c8f403dd2fd5
  48. 48
    Flynn, P. J.; Reece, R. J. Activation of Transcription by Metabolic Intermediates of the Pyrimidine Biosynthetic Pathway. Mol. Cell. Biol. 1999, 19, 882– 888,  DOI: 10.1128/MCB.19.1.882
    Google Scholar
    48
    Activation of transcription by metabolic intermediates of the pyrimidine biosynthetic pathway
    Flynn, Paul J.; Reece, Richard J.
    Molecular and Cellular Biology (1999), 19 (1), 882-888CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
    Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold. The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p. Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates. We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system. Transcriptional activation by Ppr1p can be obsd., however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions. The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein. Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvFyitA%253D%253D&md5=c3a936b1eda8f038270317fa526b055e
  49. 49
    Boeke, J. D.; La Croute, F.; Fink, G. R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 1984, 197, 345– 346,  DOI: 10.1007/BF00330984
    Google Scholar
    49
    A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoroorotic acid resistance
    Boeke, Jef D.; LaCroute, Francois; Fink, Gerald R.
    Molecular and General Genetics (1984), 197 (2), 345-6CODEN: MGGEAE; ISSN:0026-8925.
    Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a pos. selection. Wild-type strains of yeast (or ura3 mutant strains contg. a plasmid-borne URA3+ gene) are unable to grow on medium contg. the pyrimidine analog 5-fluoroorotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhs1elsrg%253D&md5=1fbe754fa41c9f426317388cbc7381ab
  50. 50
    Galvão, T. C.; De Lorenzo, V. Adaptation of the yeast URA3 selection system to Gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain. Appl. Environ. Microbiol. 2005, 71, 883– 892,  DOI: 10.1128/AEM.71.2.883-892.2005
    Google Scholar
    50
    Adaptation of the yeast URA3 selection system to Gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain
    Galvao, Teca Calcagno; De Lorenzo, Victor
    Applied and Environmental Microbiology (2005), 71 (2), 883-892CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
    A general procedure for efficient generation of gene knockouts in gram-neg. bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5'-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both pos. and neg. selection of the marker, and the robustness of the selection, where pyrF is assocd. with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-neg. bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsVKgtrw%253D&md5=03f764b6e6c5e72eb582e0cfcf77882b
  51. 51
    Cousineau, B.; Lawrence, S.; Smith, D.; Belfort, M. Retrotransposition of a bacterial group II intron. Nature 2000, 404, 1018– 1021,  DOI: 10.1038/35010029
    Google Scholar
    51
    Retrotransposition of a bacterial group II intron
    Cousineau, Benoit; Lawrence, Stacey; Smith, Dorie; Belfort, Marlene
    Nature (London) (2000), 404 (6781), 1018-1021CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown. To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis. The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymic activities. Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming. In retrohoming, the intron reverse splices into a cognate intronless DNA site. Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA. Here we report the first exptl. demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism. This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets. These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXjtFynu7s%253D&md5=8ce276b86062da0e7ec9423eae506dd0
  52. 52
    Golomb, M.; Chamberlin, M. Characterization of T7 specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. J. Biol. Chem. 1974, 249, 2858– 2863,  DOI: 10.1016/S0021-9258(19)42709-9
    Google Scholar
    52
    Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis
    Golomb, Miriam; Chamberlin, Michael
    Journal of Biological Chemistry (1974), 249 (9), 2858-63CODEN: JBCHA3; ISSN:0021-9258.
    The major in vitro RNA transcripts synthesized by T7 RNA polymerase with T7 and T3 DNA templates were resolved by electrophoresis on polyacrylamide gels. Six discrete size classes of T7 RNAs were found, and designated I to VI. The apparent mol. wts., estd. from their electrophoretic mobilities, were 2 × 105-5 × 106. At least 2 minor RNA species with mol. wts. >5 × 106 were also detected. The 6 major T7 RNA species were synthesized in approx. equimolar amts., with the exception of species III, which was made in ∼ twice this amt. Perhaps, species III is a mixt. of two RNAs transcribed from sep. regions of the T7 genome. Hence, the 6 major T7 RNA species are tentatively identified with 7 late transcription units on the phage chromosome, which are read with equal efficiencies. The 6 (or 7) transcription products were inititated independently with GTP at the 5' terminus, and were elongated at a rate of 230 nucleotides/s under standard in vitro conditions. When T7 RNA polymerase was used to transcribe T3 DNA, a single RNA transcript was found, with a mol. wt. similar to that of T7 RNA species III. This suggests an explanation for the reduced rate of T3 RNA synthesis by T7 RNA polymerase in vitro, and implies that T3 promoter sites read by the T3 RNA polymerase are heterogeneous in nature.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2cXksVKrs70%253D&md5=5abe8d2ccb186b1d414061a838117d69
  53. 53
    Chamberlin, M.; Ryan, T. 4 Bacteriophage DNA-Dependent RNA Polymerases. Enzymes 1982, 15, 87– 108
    Google Scholar
    53
    Bacteriophage DNA-dependent RNA polymerases
    Chamberlin, Michael J.; Ryan, T.
    (1982), 15 (Pt. B), 87-108CODEN: 25GLAS ISSN:. (Academic)
    A review with 87 refs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XltVGqsLo%253D&md5=edd42f3f5bbcbd40064d5fdb469be1e4
  54. 54
    Benedetti, I.; de Lorenzo, V.; Nikel, P. I. Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes. Metab. Eng. 2016, 33, 109– 118,  DOI: 10.1016/j.ymben.2015.11.004
    Google Scholar
    54
    Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes
    Benedetti, Ilaria; de Lorenzo, Victor; Nikel, Pablo I.
    Metabolic Engineering (2016), 33 (), 109-118CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
    Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degrdn. of c-di-GMP, thus making P. putida to form biofilms at user's will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phosphodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegrdn. Upon addn. of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and phys. forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochem. activity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFems7rL&md5=64291830c898a5f4ecfb5fef5313598c
  55. 55
    Martínez-Abarca, F.; García-Rodríguez, F. M.; Toro, N. Homing of a bacterial group II intron with an intron-encoded protein lacking a recognizable endonuclease domain. Mol. Microbiol. 2000, 35, 1405– 1412,  DOI: 10.1046/j.1365-2958.2000.01804.x
    Google Scholar
    55
    Homing of a bacterial group II intron with an intron-encoded protein lacking a recognizable endonuclease domain
    Martinez-Abarca, Francisco; Garcia-Rodriguez, Fernando M.; Toro, Nicolas
    Molecular Microbiology (2000), 35 (6), 1405-1412CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
    Rmlnt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630-Tc1 family. In contrast to many other group II introns, the intron-encoded protein (IEP) of Rmlnt1 lacks the characteristic conserved part of the Zn domain assocd. with the IEP endonuclease activity. Nevertheless, in this study, we show that Rmlnt1 is capable of inserting into a vector contg. the DNA spanning the Rmlnt1 target site from the genome of S. meliloti. Efficient homing was also obsd. in the absence of homologous recombination (RecA- strains). In addn., it is shown that Rmlnt1 is able to move to its target in a heterologous host (S. medicae). Homing of Rmlnt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11-30%. Afterwards, the remaining intronless target DNA is protected from intron invasion.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXis1Wrtro%253D&md5=7cf3687672ca684154f02972891ab74b
  56. 56
    Cook, T. B.; Rand, J. M.; Nurani, W.; Courtney, D. K.; Liu, S. A.; Pfleger, B. F. Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. J. Ind. Microbiol. Biotechnol. 2018, 45, 517– 527,  DOI: 10.1007/s10295-017-2001-5
    Google Scholar
    56
    Genetic tools for reliable gene expression and recombineering in Pseudomonas putida
    Cook, Taylor B.; Rand, Jacqueline M.; Nurani, Wasti; Courtney, Dylan K.; Liu, Sophia A.; Pfleger, Brian F.
    Journal of Industrial Microbiology & Biotechnology (2018), 45 (7), 517-527CODEN: JIMBFL; ISSN:1367-5435. (Springer)
    Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we detd. the copy nos. of several broad host range origins and found that plasmid copy nos. are significantly higher in P. putida KT2440 than in the synthetic biol. workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1GltA%253D%253D&md5=8404ada292d1376d0fa2b08c54d1863a
  57. 57
    Bagdasarian, M.; Lurz, R.; Rückert, B.; Franklin, F. C. H.; Bagdasarian, M. M.; Frey, J.; Timmis, K. N. Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSF 1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 1981, 16, 237– 247,  DOI: 10.1016/0378-1119(81)90080-9
    Google Scholar
    57
    Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas
    Bagdasarian, M.; Lurz, R.; Rueckert, B.; Franklin, F. C. H.; Bagdasarian, M. M.; Frey, J.; Timmis, K. N.
    Gene (1981), 16 (1-3), 237-47CODEN: GENED6; ISSN:0378-1119.
    Host-vector systems were developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-neg. host strains of P. aeruginosa and P. putida and new cloning vectors derived from the high-copy-no., broad-host-range plasmid RSF1010, which are stably maintained in a wide range of gram-neg. bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes; insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro λ packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biol. containment comparable to that of current Escherichia coli vector plasmids, are also described.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhsFKlsrg%253D&md5=886ab175b0c338a0ec3030fe6e38c178
  58. 58
    Jahn, M.; Vorpahl, C.; Hübschmann, T.; Harms, H.; Müller, S. Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microb. Cell Fact. 2016, 15, 211  DOI: 10.1186/s12934-016-0610-8
    Google Scholar
    58
    Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR
    Jahn, Michael; Vorpahl, Carsten; Huebschmann, Thomas; Harms, Hauke; Mueller, Susann
    Microbial Cell Factories (2016), 15 (), 211/1-211/12CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
    Plasmids are widely used for mol. cloning or prodn. of proteins in lab. and industrial settings. Const. modification has brought forth countless plasmid vectors whose characteristics in terms of av. plasmid copy no. (PCN) and stability are rarely known. The crucial factor detg. the PCN is the replication system; most replication systems in use today belong to a small no. of different classes and are available through repositories like the Std. European Vector Architecture (SEVA). In this study, the PCN was detd. in a set of seven SEVA-based expression plasmids only differing in the replication system. The av. PCN for all constructs was detd. by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, resp. The av. PCN detd. here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct mol. evidence that the av. PCN does not represent the true no. of plasmid mols. in individual cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjsFCrsbc%253D&md5=3245e40449919390aec4633a06832341
  59. 59
    Pyne, M. E.; Moo-Young, M.; Chung, D. A.; Chou, C. P. Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli. Appl. Environ. Microbiol. 2015, 81, 5103– 5114,  DOI: 10.1128/AEM.01248-15
    Google Scholar
    59
    Coupling the CRISPR/Cas9 system with lambda Red recombineering enables simplified chromosomal gene replacement in Escherichia coli
    Pyne, Michael E.; Moo-Young, Murray; Chung, Duane A.; Chou, C. Perry
    Applied and Environmental Microbiology (2015), 81 (15), 5103-5114CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
    To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome- encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient prodn. of gene replacement mutants that are both markerless and "scar"-less.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1Ojt7bP&md5=993a4d147fcb4c66368e6ec73bae3f7a
  60. 60
    Moreb, E. A.; Hoover, B.; Yaseen, A.; Valyasevi, N.; Roecker, Z.; Menacho-Melgar, R.; Lynch, M. D. Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. ACS Synth. Biol. 2017, 6, 2209– 2218,  DOI: 10.1021/acssynbio.7b00174
    Google Scholar
    60
    Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity
    Moreb, Eirik Adim; Hoover, Benjamin; Yaseen, Adam; Valyasevi, Nisakorn; Roecker, Zoe; Menacho-Melgar, Romel; Lynch, Michael D.
    ACS Synthetic Biology (2017), 6 (12), 2209-2218CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant neg. form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10,000 guides covering the E. coli chromosome. The use of dominant neg. RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2murrN&md5=70fcee7818b69824c249a54d3385fe39
  61. 61
    Tellechea-Luzardo, J.; Winterhalter, C.; Widera, P.; Kozyra, J.; De Lorenzo, V.; Krasnogor, N. Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering. ACS Synth. Biol. 2020, 9, 536– 545,  DOI: 10.1021/acssynbio.9b00400
    Google Scholar
    61
    Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering
    Tellechea-Luzardo, Jonathan; Winterhalter, Charles; Widera, Pawel; Kozyra, Jerzy; de Lorenzo, Victor; Krasnogor, Natalio
    ACS Synthetic Biology (2020), 9 (3), 536-545CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biol. engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnol. and digital technol., a deficit in software and lab. techniques diminishes the ability to make biotechnol. more agile, reproducible and transparent while, at the same time, limiting the security and safety of synthetic biol. constructs. To partially address some of these problems, this paper presents an approach for phys. linking engineered cells to their digital footprint-we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsVOntr0%253D&md5=282b32256dcc6a167abd1fb5b8484f29
  62. 62
    Tellechea-Luzardo, J.; Hobbs, L.; Velázquez, E.; Pelechova, L.; Woods, S.; de Lorenzo, V.; Krasnogor, N. Versioning Biological Cells for Trustworthy Cell Engineering. bioRxiv 2021.  DOI: 10.1101/2021.04.23.441106 .
    Google Scholar
    There is no corresponding record for this reference.
  63. 63
    Zimmerly, S.; Hausner, G.; Wu, X. C. Phylogenetic relationships among group II intron ORFs. Nucleic Acids Res. 2001, 29, 1238– 1250,  DOI: 10.1093/nar/29.5.1238
    Google Scholar
    63
    Phylogenetic relationships among group II intron ORFs
    Zimmerly, Steven; Hausner, Georg; Wu, Xu-Chu
    Nucleic Acids Research (2001), 29 (5), 1238-1250CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Group II introns are widely believed to have been ancestors of spliceosomal introns, yet little is known about their own evolutionary history. In order to address the evolution of mobile group II introns, the authors have compiled 71 open reading frames (ORFs) related to group II intron reverse transcriptases and subjected their derived amino acid sequences to phylogenetic anal. The phylogenetic tree was rooted with reverse transcriptases (RTs) of non-long terminal repeat retroelements, and the inferred phylogeny reveals two major clusters which the authors term the mitochondrial and chloroplast-like lineages. Bacterial ORFs are mainly positioned at the bases of the two lineages but with weak bootstrap support. The data give an overview of an apparently high degree of horizontal transfer of group II intron ORFs, mostly among related organisms but also between organelles and. bacteria. The Zn domain (nuclease) and YADD motif (RT active site) were lost multiple times during evolution. Differences in domain structures suggest that the oldest ORFs were concise, while the ORF in the mitochondrial lineage subsequently expanded in three locations. The data are consistent with a bacterial origin for mobile group II introns.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhvFGgsbk%253D&md5=a70b635e53fde84e69afc1f5b25a7d4f
  64. 64
    More, S. J.; Bampidis, V.; Benford, D.; Bragard, C.; Halldorsson, T. I.; Hernández-Jerez, A.; Susanne, H. B.; Koutsoumanis, K.; Machera, K.; Naegeli, H.; Nielsen, S. S.; Schlatter, J.; Schrenk, D.; Silano, V.; Turck, D.; Younes, M.; Glandorf, B.; Herman, L.; Tebbe, C.; Vlak, J.; Aguilera, J.; Schoonjans, R.; Cocconcelli, P. S. Evaluation of existing guidelines for their adequacy for the microbial characterisation and environmental risk assessment of microorganisms obtained through synthetic biology. EFSA J. 2020, 18, 1– 50
    Google Scholar
    There is no corresponding record for this reference.
  65. 65
    Horton, R. M.; Hunt, H. D.; Ho, S. N.; Pullen, J. K.; Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989, 77, 61– 68,  DOI: 10.1016/0378-1119(89)90359-4
    Google Scholar
    65
    Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension
    Horton, Robert M.; Hunt, Henry D.; Ho, Steffan N.; Pullen, Jeffrey K.; Pease, Larry R.
    Gene (1989), 77 (1), 61-8CODEN: GENED6; ISSN:0378-1119.
    Gene splicing by overlap extension is a new approach for recombining DNA mols. at precise junctions irresp. of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in sep. polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a mol. in which the original sequences are spliced together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different mouse class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over std. recombinant DNA techniques.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXktVais7k%253D&md5=40edd5de9a603f20bb901e78a3e3588d
  66. 66
    Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345,  DOI: 10.1038/nmeth.1318
    Google Scholar
    66
    Enzymatic assembly of DNA molecules up to several hundred kilobases
    Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
    Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480
  67. 67
    Gibson, D. G.; Glass, J. I.; Lartigue, C.; Noskov, V. N.; Chuang, R. Y.; Algire, M. A.; Benders, G. A.; Montague, M. G.; Ma, L.; Moodie, M. M.; Merryman, C.; Vashee, S.; Krishnakumar, R.; Assad-Garcia, N.; Andrews-Pfannkoch, C.; Denisova, E. A.; Young, L.; Qi, Z. N.; Segall-Shapiro, T. H.; Calvey, C. H.; Parmar, P. P.; Hutchison, C. A.; Smith, H. O.; Venter, J. C. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010, 329, 52– 56,  DOI: 10.1126/science.1190719
    Google Scholar
    67
    Creation of a bacterial cell controlled by a chemically synthesized genome
    Gibson, Daniel G.; Glass, John I.; Lartigue, Carole; Noskov, Vladimir N.; Chuang, Ray-Yuan; Algire, Mikkel A.; Benders, Gwynedd A.; Montague, Michael G.; Ma, Li; Moodie, Monzia M.; Merryman, Chuck; Vashee, Sanjay; Krishnakumar, Radha; Assad-Garcia, Nacyra; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Young, Lei; Qi, Zhi-Qing; Segall-Shapiro, Thomas H.; Calvey, Christopher H.; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
    Science (Washington, DC, United States) (2010), 329 (5987), 52-56CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. The complete, annotated synthetic genome sequence is deposited in GenBank/EMBL/DDBJ with accession no. CP002027.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotVeqsLg%253D&md5=f978c98f3e5c3c9b14bd4c7d8a1eecc7

Cited By

ARTICLE SECTIONS
Jump To

This article is cited by 4 publications.

  1. Lennart Schada von Borzyskowski, Helena Schulz-Mirbach, Mauricio Troncoso Castellanos, Francesca Severi, Paul A. Gómez-Coronado, Nicole Paczia, Timo Glatter, Arren Bar-Even, Steffen N. Lindner, Tobias J. Erb. Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol. Metabolic Engineering 2023, 76 , 97-109. https://doi.org/10.1016/j.ymben.2023.01.011
  2. Esteban Martínez-García, Sofía Fraile, Elena Algar, Tomás Aparicio, Elena Velázquez, Belén Calles, Huseyin Tas, Blas Blázquez, Bruno Martín, Clara Prieto, Lucas Sánchez-Sampedro, Morten H H Nørholm, Daniel C Volke, Nicolas T Wirth, Pavel Dvořák, Lorea Alejaldre, Lewis Grozinger, Matthew Crowther, Angel Goñi-Moreno, Pablo I Nikel, Juan Nogales, Víctor de Lorenzo. SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes. Nucleic Acids Research 2023, 51 (D1) , D1558-D1567. https://doi.org/10.1093/nar/gkac1059
  3. Jonathan Tellechea-Luzardo, Leanne Hobbs, Elena Velázquez, Lenka Pelechova, Simon Woods, Víctor de Lorenzo, Natalio Krasnogor. Versioning biological cells for trustworthy cell engineering. Nature Communications 2022, 13 (1) https://doi.org/10.1038/s41467-022-28350-4
  4. Elena Velázquez, Yamal Al‐Ramahi, Víctor de Lorenzo. CRISPR/Cas9‐enhanced Targetron Insertion for Delivery of Heterologous Sequences into the Genome of Gram‐Negative Bacteria. Current Protocols 2022, 2 (9) https://doi.org/10.1002/cpz1.532
Download PDF

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Diagram of targetrons and CRISPR/Cas9-mediated counterselection of insertions. The figure shows the technology developed in this article. Plasmid pSEVA-GIIi expresses the Ll.LtrB group II intron (empty or with cargo sequences cloned in the MluI site) and its IEP (LtrA) in the same transcriptional unit from the upstream promoter. The transcriptional unit is led by a retargeting region at 5′ (including exon 1 and the 5′ sequence of the Ll.LtrB intron), where three short sequences retrieved from the target gene (IBS, EBS1d, and EBS2) were engineered at given sites of the predicted transcript to secure its proper folding and retargeting (location of diagnostic primers is indicated). After transcription, the intronic RNA folds into a very conserved secondary structure and associates with LtrA to perform the splicing process from the exons. A lariat RNA is generated that remains attached to LtrA, forming a ribonucleoprotein (RNP) complex. This RNP scans DNA molecules until it finds the target site for retrohoming. Reverse splicing links covalently the intronic RNA to the sense strand of the DNA molecule, and the endonuclease (En) domain present in LtrA cleaves the antisense strand. Afterward, the retrotranscriptase (RT) domain of LtrA reverse transcribes the intronic RNA into DNA. The complete integration and synthesis of the cDNA is driven by repair mechanisms without the involvement of recombination. The incorporation of Cas9 complexes with gRNAs that recognize the insertion locus of Ll.LtrB causes the elimination of nonedited cells, as only those which incorporated the group II intron at the correct locus can survive (counterselection). IBS1: intron-binding site 1; EBS1d: exon-binding site 1-δ; EBS2: exon-binding site 2.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. SEVA plasmids encoding the Ll.LtrB group II intron and T7 RNAP work in E. coli BL21DE3 and P. putida KT2440. (A) Delivery of the Ll.LtrB intron from plasmid pSEVA421-GIIi (Km) in E. coli BL21DE3. The Ll.LtrB intron was retargeted to insert in the antisense orientation into the locus 1063 of the lacZ gene so that insertions would disrupt this gene, giving rise to white colonies in the presence of X-gal. Since a RAM is placed inside Ll.LtrB, kanamycin resistance was used as a way to select for intron insertion mutants (plates to the right). (B) Graph shows the number of KmR CFU normalized to 109 viable cells and classified according to the displayed phenotype in the presence of X-gal (blue colonies: lacZ+, blue bars; white colonies: lacZ– (disrupted), white hatched bars) and, also, according to the presence or absence of IPTG induction. (C) Representative colony PCR reactions to determine the correct insertion of Ll.LtrB inside the lacZ gene. Only if Ll.LtrB retrohomes, a PCR amplicon of 720 bp is generated. Blue numbers correspond to blue colonies, and black numbers correspond to white colonies used as the template material for each reaction. (D–F) Delivery of Ll.LtrB from plasmid pSEVA421-GIIi-pyrF and with the help of pSEVA131-T7RNAP in P. putida KT2440. (D) Bar plot showing the frequency of 5FOAR CFU normalized to 109 viable cells after the insertion assay. CFU are classified according to the addition or absence of IPTG during the incubation period. (E) Genuine efficiency of the insertion of Ll.LtrB. The proportion of uracil auxotrophs detected from the 5FOAR population was used as a ratio to determine the abundance of Ll.LtrB insertions in the population. (F) 5FOA counterselection was used to isolate insertion mutants that were not able to grow without uracil supplemented to plates. Colonies resistant to 5FOA but that were able to grow without uracil were used as negative controls of insertion. Two different PCR reactions are shown: (top gel) one primer annealed inside Ll.LtrB and the second annealed in the pyrF gene so that an amplicon could be only generated after intron insertion. (Bottom gel) two primers flanking the insertion locus were used so that two amplicons could be generated. The smallest fragment (380 bp) corresponds to the WT sequence, and the biggest fragment (∼1500 bp) corresponds to the insertion. The same colonies were tested in both PCR reactions. All bar graphs show the mean values (bars), standard deviation (lines), and single values (dots) of two biological replicates. WT: wild-type; Mut: insertion mutant; + control: reaction with a colony with successful insertion from a previous experiment used as a template; H2O: control PCR with no template material; UraR: colonies growing in media without uracil (no auxotrophs); UraS: colonies not growing in media without uracil (auxotrophs).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Performance of pSEVA2311-GIIi-pyrF in P. putida KT2440 and its ΔrecA derivative strain. (A) pSEVA2311-GIIi-pyrF works in P. putida KT2440 to deliver the Ll.LtrB intron into the pyrF gene through 5FOA counterselection. Left panel: Bar plot showing the frequency of 5FOAR CFUs normalized to 109 viable cells of wild-type P. putida after the insertion assay resulting from induction with various cyclohexanone concentrations (0, 0.5, 1, and 5 mM). Right panel: Genuine efficiency of insertion of Ll.LtrB. The proportion of uracil auxotrophs detected from the 5FOAR population was used as a reference to determine the abundance of Ll.LtrB insertions in each population. (B) Colony PCR reaction that used primers flanking the insertion locus was used to determine Ll.LtrB retrohoming in each concentration of cyclohexanone tested (from 0 mM in the left part of gel to 5 mM in the right part of gel). The smallest fragment (380 bp) corresponds to the wild-type sequence, while the biggest one (1500 bp) corresponds to the intron insertion in the correct location. (C, D) Functioning of pSEVA2311-GIIi-pyrF in P. putida KT2440 ΔrecA. The same experiments and analyses were done to determine the frequencies and correctness of intron insertion in the recombination-deficient strain. The gel at the bottom of (D) shows a control PCR reaction to verify recA minus genotype of the tested cells. The frequency of 5FOAR CFUs and the efficiency of insertion of Ll.LtrB in P. putida ΔrecA were determined as before. All bar graphs show the mean values (bars), standard deviation (lines), and single values (dots) of at least three biological replicates. WT, wild-type; Mut, insertion mutant; KT2440, parental strain; + control: PCR of DNA from an intron-inserted colony (from a previous experiment) used as the template; H2O: control PCR with no template material.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Assessing the size-restriction of intron-mediated delivery with CRISPR/Cas9 counterselection using luxC fragments as a cargo. (A) Schematic of the intron library generated with increasing fragment length as a cargo (from 150 up to 1050 bp) using as template the first gene of the luxCDABEG operon, luxC. The Ll.LtrB intron in pSEVA6511-GIIi (LuxN) is retargeted to insert between the nucleotides 165 and 166 of the pyrF ORF in the antisense orientation. Spacer pyrF1 recognizes the region after the insertion site (part of the recognition site is shown inside a red box). The complementary nucleotides to the PAM (5′GGG-3′) are highlighted in red and are disrupted upon intron insertion. (B) Ll.LtrB-mediated delivery of luxC fragments in P. putida KT2440 WT with CRISPR/Cas9 counterselection. Colony PCR reactions showing amplifications from colonies with Ll.LtrB::LuxØ and Ll.LtrB::Lux1 (top gel) and corresponding PCR reactions verifying the recA-plus phenotype (bottom gel). WT amplification for the recA gene is 2 kb long. (C) Ll.LtrB-mediated delivery of luxC fragments in P. putida KT2440 ΔrecA. Colony PCR reactions showing amplifications from colonies with Ll.LtrB::LuxØ to Ll.LtrB::Lux4 (top gel) and corresponding PCR reaction verifying the recA—genotype (bottom gel). Deletion of the recA gene gives an amplification of 1 kb. WT: wild-type, Mut: insertion mutant, LuxN: cargos including from LuxØ to Lux7, Ø: Ll.LtrB with no cargo, 1: Ll.LtrB with Lux1 as the cargo, 2: Ll.LtrB with Lux2 as the cargo; 3: Ll.LtrB with Lux3 as the cargo, and 4: Ll.LtrB with Lux4 as the cargo. P. putida KT2440 ΔrecA colonies with no inserted Ll.LtrB used as the negative control.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. Intron insertion frequencies with CRISPR-Cas9 counterselection in wild-type and ΔrecAP. putida KT2440 confirmed through PCR. (A) Intron insertion frequency of each cargo inserted in the genome of WT P. putida KT2440 by Ll.LtrB using 5FOA; no counterselection (control plasmid pSEVA231-CRISPR) or CRISPR/Cas9-mediated counterselection (pSEVA231-C-pyrF1). Insertion of a cargo larger than Lux1 was not detected. (B) Similarly, for P. putida KT2440 ΔrecA, insertion of a cargo larger than Lux4 was not detected. (C) Combined efficiency of targetrons and CRISPR/Cas9 counterselection. The numbers of SmR KmR CFU obtained after transforming either pSEVA231-CRISPR (control) or pSEVA231-C-pyrF1 were normalized to 109 cells. The pSEVA231-CRISPR condition was set to 100%, and the percentage of cells with pSEVA231-C-pyrF1 was calculated accordingly. Finally, the ratio of positive Ll.LtrB insertions detected in the pSEVA231-C-pyrF1 condition was multiplied individually in each replicate. The mean (bars), single values (dots), and standard deviation (lines) of two or three replicates are shown. Ø: Ll.LtrB with no cargo; 1: Ll.LtrB with Lux1 as the cargo; 2: Ll.LtrB with Lux2 as the cargo; 3: Ll.LtrB with Lux3 as the cargo; and 4: Ll.LtrB with Lux4 as the cargo.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. Application of the Ll.LtrB group II intron for delivery of specific genetic barcodes to the genome of P. putida KT2440. Organization of pSEVA6511-GIIi (B3) variants is shown with the intron retargeted toward Locus 1 (x = 37s) or Locus 2 (x = 95a). Selection of the insertion loci for Ll.LtrB::B3 is in the vicinity of the Tn7-insertion site (black triangle). Two different insertion points (gray triangles) were chosen for the insertion list generated on the Clostron website, (24) and Ll.LtrB::B3 was retargeted to both sites accordingly. The recognition site in Locus 1 (green) is located in the sense strand, while Locus 2 (orange) is present in the antisense strand of the P. putida genome. Ll.LtrB::B3 insertion would generate two different genotypes depending on the locus being targeted in each case.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 7

    Figure 7. Delivery of Ll.LtrB::B3 containing a barcode in the P. putida KT2440 genome. A first pool PCR was set to detect successful Ll.LtrB::B3 insertions in either Locus 1 (green) or Locus 2 (orange). The top gel shows the amplification found with a pool PCR using primers flanking the insertion at Locus 2. The bottom gel shows the second PCR of individual colonies from the corresponding pool to find the barcoded clone. In this case, a primer annealing inside the barcode (pbarcode universal) and another annealing inside the PP5408 gene were used. WT: wild-type, Locus 1: 36,37s insertion site, Locus 2: 94,95a insertion site. PP5408 (green gene), glmS (orange gene).

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 67 other publications.

    1. 1
      Nikel, P. I.; de Lorenzo, V. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism. Metab. Eng. 2018, 50, 142– 155,  DOI: 10.1016/j.ymben.2018.05.005
      Google Scholar
      1
      Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism
      Nikel, Pablo I.; de Lorenzo, Victor
      Metabolic Engineering (2018), 50 (), 142-155CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
      The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochem. stresses. Over the years, these properties have been capitalized biotechnol. owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biol. have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biol. systems. Much of the biotechnol. interest on P. putida stems from the distinct architecture of its central carbon metab. The native biochem. is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochem. network of P. putida (cis-metab.) has sufficed to obtain target compds. of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metab.) for making complex, new-to-Nature mols. A no. of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufg. of fine and bulk chems. in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochem. beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chem. elements into the extant biochem., as well as genomic stability and scaling-up issues.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpvVSlt7k%253D&md5=fac6e8937d2e4f3d0c26bcde01e6777c
    2. 2
      Nikel, P. I.; Martínez-García, E.; de Lorenzo, V. Biotechnological domestication of pseudomonads using synthetic biology. Nat. Rev. Microbiol. 2014, 12, 368– 379,  DOI: 10.1038/nrmicro3253
      Google Scholar
      2
      Biotechnological domestication of pseudomonads using synthetic biology
      Nikel, Pablo I.; Martinez-Garcia, Esteban; de Lorenzo, Victor
      Nature Reviews Microbiology (2014), 12 (5), 368-379CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Much of contemporary synthetic biol. research relies on the use of bacterial chassis for plugging-in and plugging-out genetic circuits and new-to-nature functionalities. However, the microorganisms that are the easiest to manipulate in the lab. are often suboptimal for downstream industrial applications, which can involve physicochem. stress and harsh operating conditions. In this Review, we advocate the use of environmental Pseudomonas strains as model organisms that are pre-endowed with the metabolic, physiol. and stress-endurance traits that are demanded by current and future synthetic biol. and biotechnol. needs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlahtL8%253D&md5=d2cc342c2b17f5631572b664f5513acb
    3. 3
      Kampers, L. F. C.; Volkers, R. J. M.; Martins Dos Santos, V. A. P. Pseudomonas putida KT2440 is HV1 certified, not GRAS. Microb. Biotechnol. 2019, 12, 845– 848,  DOI: 10.1111/1751-7915.13443
      Google Scholar
      3
      Pseudomonas putida KT2440 is HV1 certified, not GRAS
      Kampers Linde F C; Volkers Rita J M; Martins Dos Santos Vitor A P; Martins Dos Santos Vitor A P
      Microbial biotechnology (2019), 12 (5), 845-848 ISSN:.
      Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M3msleqsA%253D%253D&md5=01a803206ea94a4286be8ab8a039549c
    4. 4
      Kim, J.; Park, W. Oxidative stress response in Pseudomonas putida. Appl. Microbiol. Biotechnol. 2014, 98, 6933– 6946,  DOI: 10.1007/s00253-014-5883-4
      Google Scholar
      4
      Oxidative stress response in Pseudomonas putida
      Kim, Jisun; Park, Woojun
      Applied Microbiology and Biotechnology (2014), 98 (16), 6933-6946CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      A review. Pseudomonas putida is widely distributed in nature and is capable of degrading various org. compds. due to its high metabolic versatility. The survival capacity of P. putida stems from its frequent exposure to various endogenous and exogenous oxidative stresses. Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents existing in various niches. ROS could facilitate the evolution of bacteria by mutating genomes. Aerobic bacteria maintain defense mechanisms against oxidative stress throughout their evolution. To overcome the detrimental effects of oxidative stress, P. putida has developed defensive cellular systems involving induction of stress-sensing proteins and detoxification enzymes as well as regulation of oxidative stress response networks. Genetic responses to oxidative stress in P. putida differ markedly from those obsd. in Escherichia coli and Salmonella spp. Two major redox-sensing transcriptional regulators, SoxR and OxyR, are present and functional in the genome of P. putida. However, the novel regulators FinR and HexR control many genes belonging to the E. coli SoxR regulon. Oxidative stress can be generated by exposure to antibiotics, and iron homeostasis in P. putida is crucial for bacterial cell survival during treatment with antibiotics. This review highlights and summarizes current knowledge of oxidative stress in P. putida, as a model soil bacterium, together with recent studies from mol. genetics perspectives.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVamtLbN&md5=de993728cab8409fdd59ed5c00fcb724
    5. 5
      Nikel, P. I.; Fuhrer, T.; Chavarría, M.; Sánchez-Pascuala, A.; Sauer, U.; de Lorenzo, V. Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress. ISME J. 2021, 15, 1751– 1766,  DOI: 10.1038/s41396-020-00884-9
      Google Scholar
      5
      Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress
      Nikel, Pablo I.; Fuhrer, Tobias; Chavarria, Max; Sanchez-Pascuala, Alberto; Sauer, Uwe; de Lorenzo, Victor
      ISME Journal (2021), 15 (6), 1751-1766CODEN: IJSOCF; ISSN:1751-7362. (Nature Portfolio)
      As a frequent inhabitant of sites polluted with toxic chems., the soil bacterium and plant-root colonizer Pseudomonas putida can tolerate high levels of endogenous and exogenous oxidative stress. Yet, the ultimate reason of such phenotypic property remains largely unknown. To shed light on this question, metabolic network-wide routes for NADPH generation-the metabolic currency that fuels redox-stress quenching mechanisms-were inspected when P. putida KT2440 was challenged with a sub-lethal H2O2 dose as a proxy of oxidative conditions. 13C-tracer expts., metabolomics, and flux anal., together with the assessment of physiol. parameters and measurement of enzymic activities, revealed a substantial flux reconfiguration in oxidative environments. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidn., and the cyclic operation of the pentose phosphate pathway led to significant NADPH-forming fluxes, exceeding biosynthetic demands by ∼50%. The resulting NADPH surplus, in turn, fueled the glutathione system for H2O2 redn. These properties not only account for the tolerance of P. putida to environmental insults-some of which end up in the formation of reactive oxygen species-but they also highlight the value of this bacterial host as a platform for environmental bioremediation and metabolic engineering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVSisLc%253D&md5=31d1780e11c16b4bbdbf09c008f4324d
    6. 6
      Liao, J. C.; Mi, L.; Pontrelli, S.; Luo, S. Fuelling the future: Microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 2016, 14, 288– 304,  DOI: 10.1038/nrmicro.2016.32
      Google Scholar
      6
      Fuelling the future: microbial engineering for the production of sustainable biofuels
      Liao, James C.; Mi, Luo; Pontrelli, Sammy; Luo, Shanshan
      Nature Reviews Microbiology (2016), 14 (5), 288-304CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one soln. is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the prodn. of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compds. and how to optimize biofuel prodn. by engineering fuel pathways and central metab.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XltVaqsbk%253D&md5=fcd67bab889558ff6f918a5d70a9d6ec
    7. 7
      Jiménez, J. I.; Miñambres, B.; García, J. L.; Díaz, E. Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environ. Microbiol. 2002, 4, 824– 841,  DOI: 10.1046/j.1462-2920.2002.00370.x
      Google Scholar
      7
      Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440
      Jimenez, Jose Ignacio; Minambres, Baltasar; Garcia, Jose Luis; Diaz, Eduardo
      Environmental Microbiology (2002), 4 (12), 824-841CODEN: ENMIFM; ISSN:1462-2912. (Blackwell Science Ltd.)
      Anal. of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural arom. compds. and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central arom. intermediates, i.e., the protocatechuate (pca genes) and catechol (cat genes) branches of the β-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two addnl. gene clusters that might be involved in the catabolism of N-heterocyclic arom. compds. (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compds. (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually assocd. with the gene clusters, a supraoperonic clustering of catabolic genes that channel different arom. compds. into a common central pathway (catabolic island) was not obsd. in P. putida KT2440. The global view on the mineralization of arom. compds. by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegrdn./biotransformation processes, and reveals this bacterium as a useful model system for studying biochem., genetic, evolutionary and ecol. aspects of the catabolism of arom. compds.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvVGrtg%253D%253D&md5=1922f12af80e022107400b58fa47f419
    8. 8
      Ramos, J. L.; Duque, E.; Huertas, M. J.; Haidour, A. Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons. J. Bacteriol. 1995, 177, 3911– 3916,  DOI: 10.1128/jb.177.14.3911-3916.1995
      Google Scholar
      8
      Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons
      Ramos, Juan L.; Duque, Estrella; Huertas, Maria-Jose; Haidour, Ali
      Journal of Bacteriology (1995), 177 (14), 3911-16CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to org. solvents whose log Pow (octanol/water partition coeff.) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg2+ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concns. of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarog. studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolize toluene. Although the strain also thrived in high concns. of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmvV2gsb0%253D&md5=92a84eea5b4f2a8abba19a91b534c80b
    9. 9
      Martínez, I.; Mohamed, M. E. S.; Rozas, D.; García, J. L.; Díaz, E. Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds. Metab. Eng. 2016, 35, 46– 54,  DOI: 10.1016/j.ymben.2016.01.005
      Google Scholar
      9
      Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds
      Martinez, Igor; Mohamed, Magdy El-Said; Rozas, Daniel; Garcia, Jose Luis; Diaz, Eduardo
      Metabolic Engineering (2016), 35 (), 46-54CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
      The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of arom. heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compd. dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) mol. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial "chassis" for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative - to the previously constructed recombinant dsz cassettes - for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and once secreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient prodn. of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlalu70%253D&md5=6021bcdce6cb854cabf99a30150e080a
    10. 10
      Martínez-García, E.; Nikel, P. I.; Aparicio, T.; de Lorenzo, V. Pseudomonas 2.0: Genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb. Cell Fact. 2014, 13, 159  DOI: 10.1186/s12934-014-0159-3
      Google Scholar
      10
      Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression
      Martinez-Garcia, Esteban; Nikel, Pablo I.; Aparicio, Tomas; de Lorenzo, Victor
      Microbial Cell Factories (2014), 13 (), 159/1-159/33CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
      Background: Because of its adaptability to sites polluted with toxic chems., the model soil bacterium Pseudomonas putida is naturally endowed with a no. of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a deriv. strain in which the genome has been heavily edited in order to construct a robust microbial cell factory. Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiol. vigour. Conclusion: The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches (e.g., increasing the promoter strength in the DNA constructs of interest).
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1Oqt7g%253D&md5=dfebf084cef553ec5d6526f9fbbd504f
    11. 11
      Lieder, S.; Nikel, P. I.; de Lorenzo, V.; Takors, R. Genome reduction boosts heterologous gene expression in Pseudomonas putida. Microb. Cell Fact. 2015, 14, 23  DOI: 10.1186/s12934-015-0207-7
      Google Scholar
      11
      Genome reduction boosts heterologous gene expression in Pseudomonas putida
      Lieder Sarah; Takors Ralf; Nikel Pablo I; de Lorenzo Victor
      Microbial cell factories (2015), 14 (), 23 ISSN:.
      BACKGROUND: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome). RESULTS: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440. CONCLUSIONS: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MjlvVeqtA%253D%253D&md5=1b3829a02ebcccfb6bd3f5dfc0299255
    12. 12
      Silva-Rocha, R.; Martínez-García, E.; Calles, B.; Chavarría, M.; Arce-Rodríguez, A.; De Las Heras, A.; Páez-Espino, A. D.; Durante-Rodríguez, G.; Kim, J.; Nikel, P. I.; Platero, R.; De Lorenzo, V. The Standard European Vector Architecture (SEVA): A coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 2013, 41, D666– D675,  DOI: 10.1093/nar/gks1119
      Google Scholar
      12
      The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes
      Silva-Rocha, Rafael; Martinez-Garcia, Esteban; Calles, Belen; Chavarria, Max; Arce-Rodriguez, Alejandro; de las Heras, Aitor; Paez-Espino, A. David; Durante-Rodriguez, Gonzalo; Kim, Juhyun; Nikel, Pablo I.; Platero, Raul; de Lorenzo, Victor
      Nucleic Acids Research (2013), 41 (D1), D666-D675CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The Std. European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical lab. strains. Under the SEVA std., every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with phys. connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvV2ktrzF&md5=3ddd60ae5099d577aa6b688813a5605b
    13. 13
      Martínez-García, E.; de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: Analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 2011, 13, 2702– 2716,  DOI: 10.1111/j.1462-2920.2011.02538.x
      Google Scholar
      13
      Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440
      Martinez-Garcia, Esteban; de Lorenzo, Victor
      Environmental Microbiology (2011), 13 (10), 2702-2716CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
      The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, the authors employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-neg. bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation expts. revealed that the procedure did not increase mutation rates - perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand the authors made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to det. various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Anal. of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-neg. microorganisms.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVKhtbfP&md5=8a2204c9c029721125df30a7c2ac80e2
    14. 14
      Martínez-García, E.; De Lorenzo, V. Transposon-based and plasmid-based genetic tools for editing genomes of Gram-negative bacteria. Methods Mol. Biol. 2012, 813, 267– 283
      Google Scholar
      14
      Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria
      Martinez-Garcia, Esteban; de Lorenzo, Victor
      Methods in Molecular Biology (New York, NY, United States) (2012), 813 (Synthetic Gene Networks), 267-283CODEN: MMBIED; ISSN:1064-3745. (Springer)
      A good part of the contemporary synthetic biol. agenda aims at reprogramming microorganisms to enhance existing functions and/or perform new tasks. Moreover, the functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavors rely on the availability of genetic tools to successfully modify a la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for the prodn. of directed mutants and allelic replacements, random mutant libraries to discover new functions, and means to stably implant larger genetic networks into the genome of specific hosts. The list of gram-neg. species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available mol. techniques to do so is very limited beyond Escherichia coli. In this chapter, utilization of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of gram-neg. microorganisms.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Sqs7jK&md5=d8dc7fef841f41065b1e0c236b3339d8
    15. 15
      Martínez-García, E.; Aparicio, T.; de Lorenzo, V.; Nikel, P. I. New transposon tools tailored for metabolic engineering of Gram-negative microbial cell factories. Front. Bioeng. Biotechnol. 2014, 2, 46  DOI: 10.3389/fbioe.2014.00046
      Google Scholar
      15
      New transposon tools tailored for metabolic engineering of gram-negative microbial cell factories
      Martinez-Garcia Esteban; Aparicio Tomas; de Lorenzo Victor; Nikel Pablo I
      Frontiers in bioengineering and biotechnology (2014), 2 (), 46 ISSN:2296-4185.
      Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. A la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3nsVOrtw%253D%253D&md5=e535341c401daaac2882e3d09ba452b3
    16. 16
      Aparicio, T.; Nyerges, A.; Martínez-García, E.; de Lorenzo, V. High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering. iScience 2020, 23, 100946  DOI: 10.1016/j.isci.2020.100946
      Google Scholar
      16
      High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering
      Aparicio, Tomas; Nyerges, Akos; Martinez-Garcia, Esteban; de Lorenzo, Victor
      iScience (2020), 23 (3), 100946CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
      Application of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnol. relevant strain EM42 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutLPPE36K was designed under the control of the PL/cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments-e.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering of P. putida, as required for metabolic reprogramming of this important synthetic biol. chassis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsl2mtLg%253D&md5=7b4bae6ce59f76035cfa133e40ac0be5
    17. 17
      Martin-Pascual, M.; Batianis, C.; Bruinsma, L.; Asin-Garcia, E.; Garcia-Morales, L.; Weusthuis, R. A.; van Kranenburg, R.; Martins Dos Santos, V. A. P. A navigation guide of synthetic biology tools for Pseudomonas putida. Biotechnol. Adv. 2021, 49, 107732  DOI: 10.1016/j.biotechadv.2021.107732
      Google Scholar
      17
      A navigation guide of synthetic biology tools for Pseudomonas putida
      Martin-Pascual, Maria; Batianis, Christos; Bruinsma, Lyon; Asin-Garcia, Enrique; Garcia-Morales, Luis; Weusthuis, Ruud A.; van Kranenburg, Richard; Martins dos Santos, Vitor A. P.
      Biotechnology Advances (2021), 49 (), 107732CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)
      Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnol., owing to its remarkable metabolic versatility and ability to sustain difficult redox reactions and operational stresses, among other attractive characteristics. A wealth of genetic and in silico tools have been developed to enable the unravelling of its physiol. and improvement of its performance. However, the rise of this microbe as a promising platform for biotechnol. applications has resulted in diversification of tools and methods rather than standardization and convergence. As a consequence, multiple tools for the same purpose have been generated, while most of them have not been embraced by the scientific community, which has led to compartmentalization and inefficient use of resources. Inspired by this and by the substantial increase in popularity of P. putida, we aim herein to bring together and assess all currently available (wet and dry) synthetic biol. tools specific for this microbe, focusing on the last 5 years. We provide information on the principles, functionality, advantages and limitations, with special focus on their use in metabolic engineering. Addnl., we compare the tool portfolio for P. putida with those for other bacterial chassis and discuss potential future directions for tool development. Therefore, this review is intended as a ref. guide for experts and new 'users' of this promising chassis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1yrt7bL&md5=3012891782213362ce45205f20b9ddfe
    18. 18
      Mörl, M.; Niemer, I.; Schmelzer, C. New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates. Cell 1992, 70, 803– 810,  DOI: 10.1016/0092-8674(92)90313-2
      Google Scholar
      18
      New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates
      Morl M; Niemer I; Schmelzer C
      Cell (1992), 70 (5), 803-10 ISSN:0092-8674.
      Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate. These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK38zos1Gquw%253D%253D&md5=0de88e6dcdb98d1f7c6c68cdac24d25e
    19. 19
      Lazowska, J.; Meunier, B.; Macadre, C. Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers. EMBO J. 1994, 13, 4963– 4972,  DOI: 10.1002/j.1460-2075.1994.tb06823.x
      Google Scholar
      19
      Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers
      Lazowska, Jaga; Meunier, Brigitte; Macadre, Catherine
      EMBO Journal (1994), 13 (20), 4963-72CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
      Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COX1 gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was obsd. to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behavior of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhvF2hsrY%253D&md5=c7313b7d97832fa83b5ed5c1ba63683b
    20. 20
      Saldanha, R.; Chen, B.; Wank, H.; Matsuura, M.; Edwards, J.; Lambowitz, A. M. RNA and protein catalysis in group II intron splicing and mobility reactions using purified components. Biochemistry 1999, 38, 9069– 9083,  DOI: 10.1021/bi982799l
      Google Scholar
      20
      RNA and Protein Catalysis in Group II Intron Splicing and Mobility Reactions Using Purified Components
      Saldanha, Roland; Chen, Bing; Wank, Herbert; Matsuura, Manabu; Edwards, Judy; Lambowitz, Alan M.
      Biochemistry (1999), 38 (28), 9069-9083CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles contg. only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed anal. of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a Kd of ≤0.12 pM at 30°. This binding occurs in a rapid bimol. reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochem. anal. of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVCqtro%253D&md5=2136004d61355a78d3ad72c72f646545
    21. 21
      Matsuura, M.; Noah, J. W.; Lambowitz, A. M. Mechanism of maturase-promoted group II intron splicing. EMBO J. 2001, 20, 7259– 7270,  DOI: 10.1093/emboj/20.24.7259
      Google Scholar
      21
      Mechanism of maturase-promoted group II intron splicing
      Matsuura, Manabu; Noah, James W.; Lambowitz, Alan M.
      EMBO Journal (2001), 20 (24), 7259-7270CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
      Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chem. modification expts. that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyneratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsF2h&md5=18a1b077ef7c4155b12d2fc0bef65540
    22. 22
      Lambowitz, A. M.; Zimmerly, S. Group II introns: Mobile ribozymes that invade DNA. Cold Spring Harbor Perspect. Biol. 2011, 3, a003616  DOI: 10.1101/cshperspect.a003616
      Google Scholar
      There is no corresponding record for this reference.
    23. 23
      Perutka, J.; Wang, W.; Goerlitz, D.; Lambowitz, A. M. Use of Computer-designed Group II Introns to Disrupt Escherichia coli DExH/D-box Protein and DNA Helicase Genes. J. Mol. Biol. 2004, 336, 421– 439,  DOI: 10.1016/j.jmb.2003.12.009
      Google Scholar
      23
      Use of Computer-designed Group II Introns to Disrupt Escherichia coli DExH/D-box Protein and DNA Helicase Genes
      Perutka, Jiri; Wang, Wenjun; Goerlitz, David; Lambowitz, Alan M.
      Journal of Molecular Biology (2004), 336 (2), 421-439CODEN: JMOBAK; ISSN:0022-2836. (Elsevier)
      Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the assocd. intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small no. of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E. coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational anal. provides new insight into group II intron target site recognition, and the set of E. coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntVKrsw%253D%253D&md5=d7cde4aede8256f94d4df7eeed98ed2b
    24. 24
      Heap, J. T.; Pennington, O. J.; Cartman, S. T.; Carter, G. P.; Minton, N. P. The ClosTron: A universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods 2007, 70, 452– 464,  DOI: 10.1016/j.mimet.2007.05.021
      Google Scholar
      24
      The ClosTron: A universal gene knock-out system for the genus Clostridium
      Heap, John T.; Pennington, Oliver J.; Cartman, Stephen T.; Carter, Glen P.; Minton, Nigel P.
      Journal of Microbiological Methods (2007), 70 (3), 452-464CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)
      Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the no. of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpsFKjsLk%253D&md5=5ad07920edc8de3b8f622522223016aa
    25. 25
      Akhtar, P.; Khan, S. A. Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis. Plasmid 2012, 67, 111– 117,  DOI: 10.1016/j.plasmid.2011.12.012
      Google Scholar
      25
      Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis
      Akhtar, Parvez; Khan, Saleem A.
      Plasmid (2012), 67 (2), 111-117CODEN: PLSMDX; ISSN:0147-619X. (Elsevier)
      The large pXO1 plasmid (181.6 kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two sep. regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivs. contg. only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy no. anal. showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xks12jtrY%253D&md5=e29997ac16445fd79197fc6dbd55ccb6
    26. 26
      Frazier, C. L.; San Filippo, J.; Lambowitz, A. M.; Mills, D. A. Genetic manipulation of Lactococcus lactis by using targeted group II introns: Generation of stable insertions without selection. Appl. Environ. Microbiol. 2003, 69, 1121– 1128,  DOI: 10.1128/AEM.69.2.1121-1128.2003
      Google Scholar
      26
      Genetic manipulation of Lactococcus lactis by using targeted group II introns: Generation of stable insertions without selection
      Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.
      Applied and Environmental Microbiology (2003), 69 (2), 1121-1128CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      Despite their com. importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to sep. deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtF2it7w%253D&md5=70f040e4515aded320ec667e68c53cce
    27. 27
      Plante, I.; Cousineau, B. Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron. RNA 2006, 12, 1980– 1992,  DOI: 10.1261/rna.193306
      Google Scholar
      27
      Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron
      Plante, Isabelle; Cousineau, Benoit
      RNA (2006), 12 (11), 1980-1992CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      The Ll.LtrB intron, from the low G+C gram-pos. bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addn. of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant redn. in lariat accumulation in L. lactis. This suggests that cargo-contg. Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-contg. Ll.LtrB variants that are mobile.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFygsL%252FF&md5=12308f25b4b51b486da6ce6398af0b73
    28. 28
      Rawsthorne, H.; Turner, K. N.; Mills, D. A. Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl. Environ. Microbiol. 2006, 72, 6088– 6093,  DOI: 10.1128/AEM.02992-05
      Google Scholar
      28
      Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences
      Rawsthorne, Helen; Turner, Kevin N.; Mills, David A.
      Applied and Environmental Microbiology (2006), 72 (9), 6088-6093CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here the authors describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, resp., were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the resp. tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy no. of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same no. of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was obsd. from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addn., the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVKjt70%253D&md5=c1d1e67be17b38c6d7a699b3b11ca654
    29. 29
      Yao, J.; Zhong, J.; Fang, Y.; Geisinger, E.; Novick, R. P.; Lambowitz, A. M. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA 2006, 12, 1271– 1281,  DOI: 10.1261/rna.68706
      Google Scholar
      29
      Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing
      Yao, Jun; Zhong, Jin; Fang, Yuan; Geisinger, Edward; Novick, Richard P.; Lambowitz, Alan M.
      RNA (2006), 12 (7), 1271-1281CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivs. of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a deriv. of pCN37 with a temp.-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the prodn. of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temp.-sensitive, this method yields a conditional hsa disruptant that grows at 32° but not 43°. The temp. sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temp. sensitivity of group II intron splicing could limit the temp. range of an organism contg. a group II intron inserted in an essential gene.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xms1Cqu78%253D&md5=4e089632c8b7390ccce87c3513cbe723
    30. 30
      Karberg, M.; Guo, H.; Zhong, J.; Coon, R.; Perutka, J.; Lambowitz, A. M. Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat. Biotechnol. 2001, 19, 1162– 1167,  DOI: 10.1038/nbt1201-1162
      Google Scholar
      30
      Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria
      Karberg, Michael; Guo, Huatao; Zhong, Jin; Coon, Robert; Perutka, Jiri; Lambowitz, Alan M.
      Nature Biotechnology (2001), 19 (12), 1162-1167CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here the authors show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptV2qsrw%253D&md5=3dad957336fc3ad3a37f4316d019bf4a
    31. 31
      García-Rodríguez, F. M.; Hernańdez-Gutiérrez, T.; Diáz-Prado, V.; Toro, N. Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting. RNA Biol. 2014, 11, 391– 401,  DOI: 10.4161/rna.28373
      Google Scholar
      31
      Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting
      Garcia-Rodriguez Fernando M; Hernandez-Gutierrez Teresa; Diaz-Prado Vanessa; Toro Nicolas
      RNA biology (2014), 11 (4), 391-401 ISSN:.
      Gene-targeting vectors derived from mobile group II introns capable of forming a ribonucleoprotein (RNP) complex containing excised intron lariat RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT), maturase, and endonuclease (En) activities have been described. RmInt1 is an efficient mobile group II intron with an IEP lacking the En domain. We performed a comprehensive study of the rules governing RmInt1 target site recognition based on selection experiments with donor and recipient plasmid libraries, with randomization of the elements of the intron RNA involved in target recognition and the wild-type target site. The data obtained were used to develop a computer algorithm for identifying potential RmInt1 targets in any DNA sequence. Using this algorithm, we modified RmInt1 for the efficient recognition of DNA target sites at different locations in the Sinorhizobium meliloti chromosome. The retargeted RmInt1 integrated efficiently into the chromosome, regardless of the location of the target gene. Our results suggest that RmInt1 could be efficiently adapted for gene targeting.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2crlsFehsA%253D%253D&md5=49269dde7cb61dfd2adf91c2aa0d3971
    32. 32
      Cousineau, B.; Smith, D.; Lawrence-Cavanagh, S.; Mueller, J. E.; Yang, J.; Mills, D.; Manias, D.; Dunny, G.; Lambowitz, A. M.; Belfort, M. Retrohoming of a bacterial group II intron: Mobility via complete reverse splicing, independent of homologous DNA recombination. Cell 1998, 94, 451– 462,  DOI: 10.1016/S0092-8674(00)81586-X
      Google Scholar
      32
      Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination
      Cousineau, Benoit; Smith, Dorie; Lawrence-Cavanagh, Stacey; Mueller, John E.; Yang, Jian; Mills, David; Dunny, Gary; Lambowitz, Alan M.; Belfort, Marlene
      Cell (Cambridge, Massachusetts) (1998), 94 (4), 451-462CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The mobile group II intron of Lactococcus lactis, LI.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that LI.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homol. and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, LI.LtrB has many features of retrotransposons, with practical and evolutionary implications.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlslOktb8%253D&md5=ebbed8461cc1dadf9c870f01354bac6d
    33. 33
      Velázquez, E.; Lorenzo, V. D.; Al-Ramahi, Y. Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery. ACS Synth. Biol. 2019, 8, 2186– 2193,  DOI: 10.1021/acssynbio.9b00293
      Google Scholar
      33
      Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery
      Velazquez, Elena; Lorenzo, Victor de; Al-Ramahi, Yamal
      ACS Synthetic Biology (2019), 8 (9), 2186-2193CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Group II introns were developed time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-pos. and Gram-neg. bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of addnl. genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counter-selection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed proved that directing CRISPR/Cas9 towards the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counter selection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2ntbzN&md5=f06edd98e918993b500d50caa1978a9c
    34. 34
      Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007, 315, 1709– 1712,  DOI: 10.1126/science.1138140
      Google Scholar
      34
      CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
      Barrangou, Rodolphe; Fremaux, Christophe; Deveau, Helene; Richards, Melissa; Boyaval, Patrick; Moineau, Sylvain; Romero, Dennis A.; Horvath, Philippe
      Science (Washington, DC, United States) (2007), 315 (5819), 1709-1712CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addn. of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with assocd. cas genes, provided resistance against phages, and resistance specificity is detd. by spacer-phage sequence similarity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtlWntb8%253D&md5=b88c0100d2c0469213afda20e47c39cd
    35. 35
      Jiang, W.; Bikard, D.; Cox, D.; Zhang, F.; Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 2013, 31, 233– 239,  DOI: 10.1038/nbt.2508
      Google Scholar
      35
      RNA-guided editing of bacterial genomes using CRISPR-Cas systems
      Jiang, Wenyan; Bikard, David; Cox, David; Zhang, Feng; Marraffini, Luciano A.
      Nature Biotechnology (2013), 31 (3), 233-239CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-assocd. Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFCkurY%253D&md5=ea3bbfda133b51e71b114de2c9ceabfd
    36. 36
      Aparicio, T.; de Lorenzo, V.; Martínez-García, E. CRISPR/Cas9-Based Counterselection Boosts Recombineering Efficiency in Pseudomonas putida. Biotechnol. J. 2018, 13, 1700161  DOI: 10.1002/biot.201700161
      Google Scholar
      There is no corresponding record for this reference.
    37. 37
      Benedetti, I.; Nikel, P. I.; de Lorenzo, V. Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria. Data Brief 2016, 6, 738– 744,  DOI: 10.1016/j.dib.2016.01.022
      Google Scholar
      37
      Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria
      Benedetti Ilaria; Nikel Pablo I; de Lorenzo Victor
      Data in brief (2016), 6 (), 738-44 ISSN:2352-3409.
      Engineering of robust microbial cell factories requires the use of dedicated genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We have edited and formatted the ChnR/P chnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The proposed compositional standard was thoroughly verified with a monomeric and superfolder green fluorescent protein (msf•GFP) in Escherichia coli. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Data in this article are related to the research paper "Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes" [1].
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jgt1Onug%253D%253D&md5=5c2853ffbcc263263df26ad415e9484a
    38. 38
      Yao, J.; Lambowitz, A. M. Gene targeting in Gram-negative bacteria by use of a mobile group II intron (“targetron”) expressed from a broad-host-range vector. Appl. Environ. Microbiol. 2007, 73, 2735– 2743,  DOI: 10.1128/AEM.02829-06
      Google Scholar
      38
      Gene targeting in gram-negative bacteria by use of a mobile group II intron ("targetron") expressed from a broad-host-range vector
      Yao, Jun; Lambowitz, Alan M.
      Applied and Environmental Microbiology (2007), 73 (8), 2735-2743CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector contg. an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-neg. bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvFeqsbc%253D&md5=136a1d18e1873775be03c8940174d9fe
    39. 39
      Martínez-García, E.; Aparicio, T.; Goñi-Moreno, A.; Fraile, S.; De Lorenzo, V. SEVA 2.0: An update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic Acids Res. 2015, 43, D1183– D1189,  DOI: 10.1093/nar/gku1114
      Google Scholar
      39
      SEVA 2.0: an update of the standard European vector architecture for de-/re-construction of bacterial functionalities
      Martinez-Garcia, Esteban; Aparicio, Tomas; Goni-Moreno, Angel; Fraile, Sofia; de Lorenzo, Victor
      Nucleic Acids Research (2015), 43 (D1), D1183-D1189CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A review. The Std. European Vector Architecture 2.0 database (SEVA-DB 2.0) is an improved and expanded version of the platform released in 2013 aimed at assisting the choice of optimal genetic tools for deconstructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA std. facilitates combinations of functional DNA segments that ease both the anal. and the engineering of diverse Gram-neg. bacteria for fundamental or biotechnol. purposes. The large no. of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biol. Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biol. communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVymtLfL&md5=8152ca1366af4c0fe1d99435b137bbcb
    40. 40
      Martínez-García, E.; Goñi-Moreno, A.; Bartley, B.; McLaughlin, J.; Sánchez-Sampedro, L.; Pascual Del Pozo, H.; Prieto Hernández, C.; Marletta, A. S.; De Lucrezia, D.; Sánchez-Fernández, G.; Fraile, S.; De Lorenzo, V. SEVA 3.0: An update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts. Nucleic Acids Res. 2020, 48, D1164– D1170,  DOI: 10.1093/nar/gkz1024
      Google Scholar
      40
      SEVA 3.0: an update of the standard European vector architecture for enabling portability of genetic constructs among diverse bacterial hosts
      Martinez-Garcia, Esteban; Goni-Moreno, Angel; Bartley, Bryan; McLaughlin, James; Sanchez-Sampedro, Lucas; del Pozo, Hector Pascual; Hernandez, Clara Prieto; Marletta, Ada Serena; De Lucrezia, Davide; Sanchez-Fernandez, Guzman; Fraile, Sofia; de Lorenzo, Victor
      Nucleic Acids Research (2020), 48 (D1), D1164-D1170CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. The Std. European Vector Architecture 3.0 database is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for anal., construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several tech., computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biol. Open Language) format. This ensures interoperability with other platforms and affords simulations of their behavior under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biol. approaches towards non-std. bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnol. endeavours.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1GltrbP&md5=4f5a11f9bf0a49d09a3ff86bcfdd1252
    41. 41
      Zhong, J.; Karberg, M.; Lambowitz, A. M. Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res. 2003, 31, 1656– 1664,  DOI: 10.1093/nar/gkg248
      Google Scholar
      41
      Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker
      Zhong, Jin; Karberg, Michael; Lambowitz, Alan M.
      Nucleic Acids Research (2003), 31 (6), 1656-1664CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron contg. a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Anal. of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy no. of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXit1yks7g%253D&md5=9ddea609f3357b85ce578dbb1967c657
    42. 42
      Studier, F. W.; Moffatt, B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 1986, 189, 113– 130,  DOI: 10.1016/0022-2836(86)90385-2
      Google Scholar
      42
      Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
      Studier, F. William; Moffatt, Barbara A.
      Journal of Molecular Biology (1986), 189 (1), 113-30CODEN: JMOBAK; ISSN:0022-2836.
      A gene expression system based on phage T7 RNA polymerase [9014-24-8] was developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amt. of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for rRNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and(or) stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degrdn. Apparently, a specific mRNA produced by T7 RNA polymerase can rapidly sat. the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to >50% of the total cell protein in ≤3 h. Two ways were used to deliver active T7 RNA polymerase to the cell: (1) infection by a λ deriv. that carried gene 1; or (2) induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silently in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable. But if the target plasmid can be maintained, induction of chromosomal gene 1 can be a convenient way to produce large amts. of target RNA and(or) protein. T7 RNA polymerase seems to be capable of transcribing almost any DNA linked to a T7 promoter, so the T7 expression system should be capable of transcribing almost any gene or its complement in E. coli. Comparable T7 expression systems can be developed in other types of cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XktlKrsr4%253D&md5=3219300bc2f640fe9830c7518eb99bfc
    43. 43
      Dubendorf, J. W.; Studier, F. W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 1991, 219, 45– 59,  DOI: 10.1016/0022-2836(91)90856-2
      Google Scholar
      There is no corresponding record for this reference.
    44. 44
      Angius, F.; Ilioaia, O.; Amrani, A.; Suisse, A.; Rosset, L.; Legrand, A.; Abou-Hamdan, A.; Uzan, M.; Zito, F.; Miroux, B. A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins. Sci. Rep. 2018, 8, 8572  DOI: 10.1038/s41598-018-26668-y
      Google Scholar
      44
      A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins
      Angius Federica; Ilioaia Oana; Amrani Amira; Suisse Annabelle; Rosset Lindsay; Legrand Amelie; Abou-Hamdan Abbas; Uzan Marc; Zito Francesca; Miroux Bruno; Suisse Annabelle; Abou-Hamdan Abbas
      Scientific reports (2018), 8 (1), 8572 ISSN:.
      Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mbkt12isg%253D%253D&md5=0256960f3095dbbacbc5be9d233b3c38
    45. 45
      Ichiyanagi, K.; Beauregard, A.; Lawrence, S.; Smith, D.; Cousineau, B.; Belfort, M. Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets. Mol. Microbiol. 2002, 46, 1259– 1272,  DOI: 10.1046/j.1365-2958.2002.03226.x
      Google Scholar
      45
      Retrotransposition of the LI.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets
      Ichiyanagi, Kenji; Beauregard, Arthur; Lawrence, Stacey; Smith, Dorie; Cousineau, Benoit; Belfort, Marlene
      Molecular Microbiology (2002), 46 (5), 1259-1272CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
      Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double-stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, LI.LtrB, supported frequent use of RNA targets during retrotransposition, recent expts. with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single-stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron-encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging-strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides crit. for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging-strand template, hinting at a role for DNA replication in intron dispersal in nature.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsFGisL0%253D&md5=a588926c206e0bf19be2da6bb66a36c9
    46. 46
      Dickson, L.; Huang, H. R.; Liu, L.; Matsuura, M.; Lambowitz, A. M.; Perlman, P. S. Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13207– 13212,  DOI: 10.1073/pnas.231494498
      Google Scholar
      46
      Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites
      Dickson, Lorna; Huang, Hon-Ren; Liu, Lu; Matsuura, Manabu; Lambowitz, Alan M.; Perlman, Philip S.
      Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (23), 13207-13212CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addn. to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXosFygtrs%253D&md5=5bd9e382b0d94d8b6bb91f15232e59cc
    47. 47
      Coros, C. J.; Landthaler, M.; Piazza, C. L.; Beauregard, A.; Esposito, D.; Perutka, J.; Lambowitz, A. M.; Belfort, M. Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment. Mol. Microbiol. 2005, 56, 509– 524,  DOI: 10.1111/j.1365-2958.2005.04554.x
      Google Scholar
      47
      Retrotransposition strategies of the lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment
      Coros, Colin J.; Landthaler, Markus; Piazza, Carol Lyn; Beauregard, Arthur; Esposito, Donna; Perutka, Jiri; Lambowitz, Alan M.; Belfort, Marlene
      Molecular Microbiology (2005), 56 (2), 509-524CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Addnl., growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjsFWmtLs%253D&md5=d68f89a37bc9cd4bd4c9c8f403dd2fd5
    48. 48
      Flynn, P. J.; Reece, R. J. Activation of Transcription by Metabolic Intermediates of the Pyrimidine Biosynthetic Pathway. Mol. Cell. Biol. 1999, 19, 882– 888,  DOI: 10.1128/MCB.19.1.882
      Google Scholar
      48
      Activation of transcription by metabolic intermediates of the pyrimidine biosynthetic pathway
      Flynn, Paul J.; Reece, Richard J.
      Molecular and Cellular Biology (1999), 19 (1), 882-888CODEN: MCEBD4; ISSN:0270-7306. (American Society for Microbiology)
      Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold. The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p. Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates. We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system. Transcriptional activation by Ppr1p can be obsd., however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions. The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein. Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvFyitA%253D%253D&md5=c3a936b1eda8f038270317fa526b055e
    49. 49
      Boeke, J. D.; La Croute, F.; Fink, G. R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 1984, 197, 345– 346,  DOI: 10.1007/BF00330984
      Google Scholar
      49
      A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoroorotic acid resistance
      Boeke, Jef D.; LaCroute, Francois; Fink, Gerald R.
      Molecular and General Genetics (1984), 197 (2), 345-6CODEN: MGGEAE; ISSN:0026-8925.
      Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a pos. selection. Wild-type strains of yeast (or ura3 mutant strains contg. a plasmid-borne URA3+ gene) are unable to grow on medium contg. the pyrimidine analog 5-fluoroorotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhs1elsrg%253D&md5=1fbe754fa41c9f426317388cbc7381ab
    50. 50
      Galvão, T. C.; De Lorenzo, V. Adaptation of the yeast URA3 selection system to Gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain. Appl. Environ. Microbiol. 2005, 71, 883– 892,  DOI: 10.1128/AEM.71.2.883-892.2005
      Google Scholar
      50
      Adaptation of the yeast URA3 selection system to Gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain
      Galvao, Teca Calcagno; De Lorenzo, Victor
      Applied and Environmental Microbiology (2005), 71 (2), 883-892CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      A general procedure for efficient generation of gene knockouts in gram-neg. bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5'-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both pos. and neg. selection of the marker, and the robustness of the selection, where pyrF is assocd. with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-neg. bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsVKgtrw%253D&md5=03f764b6e6c5e72eb582e0cfcf77882b
    51. 51
      Cousineau, B.; Lawrence, S.; Smith, D.; Belfort, M. Retrotransposition of a bacterial group II intron. Nature 2000, 404, 1018– 1021,  DOI: 10.1038/35010029
      Google Scholar
      51
      Retrotransposition of a bacterial group II intron
      Cousineau, Benoit; Lawrence, Stacey; Smith, Dorie; Belfort, Marlene
      Nature (London) (2000), 404 (6781), 1018-1021CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown. To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis. The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymic activities. Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming. In retrohoming, the intron reverse splices into a cognate intronless DNA site. Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA. Here we report the first exptl. demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism. This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets. These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXjtFynu7s%253D&md5=8ce276b86062da0e7ec9423eae506dd0
    52. 52
      Golomb, M.; Chamberlin, M. Characterization of T7 specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. J. Biol. Chem. 1974, 249, 2858– 2863,  DOI: 10.1016/S0021-9258(19)42709-9
      Google Scholar
      52
      Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis
      Golomb, Miriam; Chamberlin, Michael
      Journal of Biological Chemistry (1974), 249 (9), 2858-63CODEN: JBCHA3; ISSN:0021-9258.
      The major in vitro RNA transcripts synthesized by T7 RNA polymerase with T7 and T3 DNA templates were resolved by electrophoresis on polyacrylamide gels. Six discrete size classes of T7 RNAs were found, and designated I to VI. The apparent mol. wts., estd. from their electrophoretic mobilities, were 2 × 105-5 × 106. At least 2 minor RNA species with mol. wts. >5 × 106 were also detected. The 6 major T7 RNA species were synthesized in approx. equimolar amts., with the exception of species III, which was made in ∼ twice this amt. Perhaps, species III is a mixt. of two RNAs transcribed from sep. regions of the T7 genome. Hence, the 6 major T7 RNA species are tentatively identified with 7 late transcription units on the phage chromosome, which are read with equal efficiencies. The 6 (or 7) transcription products were inititated independently with GTP at the 5' terminus, and were elongated at a rate of 230 nucleotides/s under standard in vitro conditions. When T7 RNA polymerase was used to transcribe T3 DNA, a single RNA transcript was found, with a mol. wt. similar to that of T7 RNA species III. This suggests an explanation for the reduced rate of T3 RNA synthesis by T7 RNA polymerase in vitro, and implies that T3 promoter sites read by the T3 RNA polymerase are heterogeneous in nature.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2cXksVKrs70%253D&md5=5abe8d2ccb186b1d414061a838117d69
    53. 53
      Chamberlin, M.; Ryan, T. 4 Bacteriophage DNA-Dependent RNA Polymerases. Enzymes 1982, 15, 87– 108
      Google Scholar
      53
      Bacteriophage DNA-dependent RNA polymerases
      Chamberlin, Michael J.; Ryan, T.
      (1982), 15 (Pt. B), 87-108CODEN: 25GLAS ISSN:. (Academic)
      A review with 87 refs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XltVGqsLo%253D&md5=edd42f3f5bbcbd40064d5fdb469be1e4
    54. 54
      Benedetti, I.; de Lorenzo, V.; Nikel, P. I. Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes. Metab. Eng. 2016, 33, 109– 118,  DOI: 10.1016/j.ymben.2015.11.004
      Google Scholar
      54
      Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes
      Benedetti, Ilaria; de Lorenzo, Victor; Nikel, Pablo I.
      Metabolic Engineering (2016), 33 (), 109-118CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
      Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degrdn. of c-di-GMP, thus making P. putida to form biofilms at user's will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phosphodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegrdn. Upon addn. of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and phys. forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochem. activity.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFems7rL&md5=64291830c898a5f4ecfb5fef5313598c
    55. 55
      Martínez-Abarca, F.; García-Rodríguez, F. M.; Toro, N. Homing of a bacterial group II intron with an intron-encoded protein lacking a recognizable endonuclease domain. Mol. Microbiol. 2000, 35, 1405– 1412,  DOI: 10.1046/j.1365-2958.2000.01804.x
      Google Scholar
      55
      Homing of a bacterial group II intron with an intron-encoded protein lacking a recognizable endonuclease domain
      Martinez-Abarca, Francisco; Garcia-Rodriguez, Fernando M.; Toro, Nicolas
      Molecular Microbiology (2000), 35 (6), 1405-1412CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
      Rmlnt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630-Tc1 family. In contrast to many other group II introns, the intron-encoded protein (IEP) of Rmlnt1 lacks the characteristic conserved part of the Zn domain assocd. with the IEP endonuclease activity. Nevertheless, in this study, we show that Rmlnt1 is capable of inserting into a vector contg. the DNA spanning the Rmlnt1 target site from the genome of S. meliloti. Efficient homing was also obsd. in the absence of homologous recombination (RecA- strains). In addn., it is shown that Rmlnt1 is able to move to its target in a heterologous host (S. medicae). Homing of Rmlnt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11-30%. Afterwards, the remaining intronless target DNA is protected from intron invasion.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXis1Wrtro%253D&md5=7cf3687672ca684154f02972891ab74b
    56. 56
      Cook, T. B.; Rand, J. M.; Nurani, W.; Courtney, D. K.; Liu, S. A.; Pfleger, B. F. Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. J. Ind. Microbiol. Biotechnol. 2018, 45, 517– 527,  DOI: 10.1007/s10295-017-2001-5
      Google Scholar
      56
      Genetic tools for reliable gene expression and recombineering in Pseudomonas putida
      Cook, Taylor B.; Rand, Jacqueline M.; Nurani, Wasti; Courtney, Dylan K.; Liu, Sophia A.; Pfleger, Brian F.
      Journal of Industrial Microbiology & Biotechnology (2018), 45 (7), 517-527CODEN: JIMBFL; ISSN:1367-5435. (Springer)
      Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we detd. the copy nos. of several broad host range origins and found that plasmid copy nos. are significantly higher in P. putida KT2440 than in the synthetic biol. workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1GltA%253D%253D&md5=8404ada292d1376d0fa2b08c54d1863a
    57. 57
      Bagdasarian, M.; Lurz, R.; Rückert, B.; Franklin, F. C. H.; Bagdasarian, M. M.; Frey, J.; Timmis, K. N. Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSF 1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 1981, 16, 237– 247,  DOI: 10.1016/0378-1119(81)90080-9
      Google Scholar
      57
      Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas
      Bagdasarian, M.; Lurz, R.; Rueckert, B.; Franklin, F. C. H.; Bagdasarian, M. M.; Frey, J.; Timmis, K. N.
      Gene (1981), 16 (1-3), 237-47CODEN: GENED6; ISSN:0378-1119.
      Host-vector systems were developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-neg. host strains of P. aeruginosa and P. putida and new cloning vectors derived from the high-copy-no., broad-host-range plasmid RSF1010, which are stably maintained in a wide range of gram-neg. bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes; insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro λ packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biol. containment comparable to that of current Escherichia coli vector plasmids, are also described.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhsFKlsrg%253D&md5=886ab175b0c338a0ec3030fe6e38c178
    58. 58
      Jahn, M.; Vorpahl, C.; Hübschmann, T.; Harms, H.; Müller, S. Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microb. Cell Fact. 2016, 15, 211  DOI: 10.1186/s12934-016-0610-8
      Google Scholar
      58
      Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR
      Jahn, Michael; Vorpahl, Carsten; Huebschmann, Thomas; Harms, Hauke; Mueller, Susann
      Microbial Cell Factories (2016), 15 (), 211/1-211/12CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
      Plasmids are widely used for mol. cloning or prodn. of proteins in lab. and industrial settings. Const. modification has brought forth countless plasmid vectors whose characteristics in terms of av. plasmid copy no. (PCN) and stability are rarely known. The crucial factor detg. the PCN is the replication system; most replication systems in use today belong to a small no. of different classes and are available through repositories like the Std. European Vector Architecture (SEVA). In this study, the PCN was detd. in a set of seven SEVA-based expression plasmids only differing in the replication system. The av. PCN for all constructs was detd. by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, resp. The av. PCN detd. here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct mol. evidence that the av. PCN does not represent the true no. of plasmid mols. in individual cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjsFCrsbc%253D&md5=3245e40449919390aec4633a06832341
    59. 59
      Pyne, M. E.; Moo-Young, M.; Chung, D. A.; Chou, C. P. Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli. Appl. Environ. Microbiol. 2015, 81, 5103– 5114,  DOI: 10.1128/AEM.01248-15
      Google Scholar
      59
      Coupling the CRISPR/Cas9 system with lambda Red recombineering enables simplified chromosomal gene replacement in Escherichia coli
      Pyne, Michael E.; Moo-Young, Murray; Chung, Duane A.; Chou, C. Perry
      Applied and Environmental Microbiology (2015), 81 (15), 5103-5114CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
      To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome- encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient prodn. of gene replacement mutants that are both markerless and "scar"-less.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1Ojt7bP&md5=993a4d147fcb4c66368e6ec73bae3f7a
    60. 60
      Moreb, E. A.; Hoover, B.; Yaseen, A.; Valyasevi, N.; Roecker, Z.; Menacho-Melgar, R.; Lynch, M. D. Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. ACS Synth. Biol. 2017, 6, 2209– 2218,  DOI: 10.1021/acssynbio.7b00174
      Google Scholar
      60
      Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity
      Moreb, Eirik Adim; Hoover, Benjamin; Yaseen, Adam; Valyasevi, Nisakorn; Roecker, Zoe; Menacho-Melgar, Romel; Lynch, Michael D.
      ACS Synthetic Biology (2017), 6 (12), 2209-2218CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant neg. form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10,000 guides covering the E. coli chromosome. The use of dominant neg. RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2murrN&md5=70fcee7818b69824c249a54d3385fe39
    61. 61
      Tellechea-Luzardo, J.; Winterhalter, C.; Widera, P.; Kozyra, J.; De Lorenzo, V.; Krasnogor, N. Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering. ACS Synth. Biol. 2020, 9, 536– 545,  DOI: 10.1021/acssynbio.9b00400
      Google Scholar
      61
      Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering
      Tellechea-Luzardo, Jonathan; Winterhalter, Charles; Widera, Pawel; Kozyra, Jerzy; de Lorenzo, Victor; Krasnogor, Natalio
      ACS Synthetic Biology (2020), 9 (3), 536-545CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biol. engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnol. and digital technol., a deficit in software and lab. techniques diminishes the ability to make biotechnol. more agile, reproducible and transparent while, at the same time, limiting the security and safety of synthetic biol. constructs. To partially address some of these problems, this paper presents an approach for phys. linking engineered cells to their digital footprint-we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsVOntr0%253D&md5=282b32256dcc6a167abd1fb5b8484f29
    62. 62
      Tellechea-Luzardo, J.; Hobbs, L.; Velázquez, E.; Pelechova, L.; Woods, S.; de Lorenzo, V.; Krasnogor, N. Versioning Biological Cells for Trustworthy Cell Engineering. bioRxiv 2021.  DOI: 10.1101/2021.04.23.441106 .
      Google Scholar
      There is no corresponding record for this reference.
    63. 63
      Zimmerly, S.; Hausner, G.; Wu, X. C. Phylogenetic relationships among group II intron ORFs. Nucleic Acids Res. 2001, 29, 1238– 1250,  DOI: 10.1093/nar/29.5.1238
      Google Scholar
      63
      Phylogenetic relationships among group II intron ORFs
      Zimmerly, Steven; Hausner, Georg; Wu, Xu-Chu
      Nucleic Acids Research (2001), 29 (5), 1238-1250CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Group II introns are widely believed to have been ancestors of spliceosomal introns, yet little is known about their own evolutionary history. In order to address the evolution of mobile group II introns, the authors have compiled 71 open reading frames (ORFs) related to group II intron reverse transcriptases and subjected their derived amino acid sequences to phylogenetic anal. The phylogenetic tree was rooted with reverse transcriptases (RTs) of non-long terminal repeat retroelements, and the inferred phylogeny reveals two major clusters which the authors term the mitochondrial and chloroplast-like lineages. Bacterial ORFs are mainly positioned at the bases of the two lineages but with weak bootstrap support. The data give an overview of an apparently high degree of horizontal transfer of group II intron ORFs, mostly among related organisms but also between organelles and. bacteria. The Zn domain (nuclease) and YADD motif (RT active site) were lost multiple times during evolution. Differences in domain structures suggest that the oldest ORFs were concise, while the ORF in the mitochondrial lineage subsequently expanded in three locations. The data are consistent with a bacterial origin for mobile group II introns.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhvFGgsbk%253D&md5=a70b635e53fde84e69afc1f5b25a7d4f
    64. 64
      More, S. J.; Bampidis, V.; Benford, D.; Bragard, C.; Halldorsson, T. I.; Hernández-Jerez, A.; Susanne, H. B.; Koutsoumanis, K.; Machera, K.; Naegeli, H.; Nielsen, S. S.; Schlatter, J.; Schrenk, D.; Silano, V.; Turck, D.; Younes, M.; Glandorf, B.; Herman, L.; Tebbe, C.; Vlak, J.; Aguilera, J.; Schoonjans, R.; Cocconcelli, P. S. Evaluation of existing guidelines for their adequacy for the microbial characterisation and environmental risk assessment of microorganisms obtained through synthetic biology. EFSA J. 2020, 18, 1– 50
      Google Scholar
      There is no corresponding record for this reference.
    65. 65
      Horton, R. M.; Hunt, H. D.; Ho, S. N.; Pullen, J. K.; Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989, 77, 61– 68,  DOI: 10.1016/0378-1119(89)90359-4
      Google Scholar
      65
      Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension
      Horton, Robert M.; Hunt, Henry D.; Ho, Steffan N.; Pullen, Jeffrey K.; Pease, Larry R.
      Gene (1989), 77 (1), 61-8CODEN: GENED6; ISSN:0378-1119.
      Gene splicing by overlap extension is a new approach for recombining DNA mols. at precise junctions irresp. of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in sep. polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a mol. in which the original sequences are spliced together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different mouse class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over std. recombinant DNA techniques.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXktVais7k%253D&md5=40edd5de9a603f20bb901e78a3e3588d
    66. 66
      Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345,  DOI: 10.1038/nmeth.1318
      Google Scholar
      66
      Enzymatic assembly of DNA molecules up to several hundred kilobases
      Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
      Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480
    67. 67
      Gibson, D. G.; Glass, J. I.; Lartigue, C.; Noskov, V. N.; Chuang, R. Y.; Algire, M. A.; Benders, G. A.; Montague, M. G.; Ma, L.; Moodie, M. M.; Merryman, C.; Vashee, S.; Krishnakumar, R.; Assad-Garcia, N.; Andrews-Pfannkoch, C.; Denisova, E. A.; Young, L.; Qi, Z. N.; Segall-Shapiro, T. H.; Calvey, C. H.; Parmar, P. P.; Hutchison, C. A.; Smith, H. O.; Venter, J. C. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010, 329, 52– 56,  DOI: 10.1126/science.1190719
      Google Scholar
      67
      Creation of a bacterial cell controlled by a chemically synthesized genome
      Gibson, Daniel G.; Glass, John I.; Lartigue, Carole; Noskov, Vladimir N.; Chuang, Ray-Yuan; Algire, Mikkel A.; Benders, Gwynedd A.; Montague, Michael G.; Ma, Li; Moodie, Monzia M.; Merryman, Chuck; Vashee, Sanjay; Krishnakumar, Radha; Assad-Garcia, Nacyra; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Young, Lei; Qi, Zhi-Qing; Segall-Shapiro, Thomas H.; Calvey, Christopher H.; Parmar, Prashanth P.; Hutchison, Clyde A., III; Smith, Hamilton O.; Venter, J. Craig
      Science (Washington, DC, United States) (2010), 329 (5987), 52-56CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. The complete, annotated synthetic genome sequence is deposited in GenBank/EMBL/DDBJ with accession no. CP002027.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotVeqsLg%253D&md5=f978c98f3e5c3c9b14bd4c7d8a1eecc7

  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.1c00199.

    • List of oligonucleotides used in this study (Supplementary Table S1); list of plasmids used in this work (Supplementary Table S2); insertion frequencies of Ll.LtrB::Lux1 and Ll.LtrB::Lux4 in P. putida KT2440 WT and ΔrecA with no CRISPR/Cas9-mediated counterselection (Supplementary Table S3); insertion frequency of Ll.LtrB::LuxN intron in P. putida KT2440 WT with 5FOA CRISPR/Cas9-mediated counterselection (Supplementary Table S4); insertion frequency of Ll.LtrB::LuxN intron in P. putida KT2440 ΔrecA with 5FOA CRISPR/Cas9-mediated counterselection (Supplementary Table S5); construction and verification of intron delivery plasmids compatible with CRISPR/Cas9-mediated counterselection (Supplementary Methods); pSEVA plasmids for the expression of the Ll.LtrB intron in a wide range of Gram-negative bacteria (Supplementary Figure S1); preliminary approach to assess size-restriction of intron-mediated delivery using luxC fragments as a cargo and 5FOA counterselection in log-phase induced cells (Supplementary Figure S2); total intron insertion frequency without differentiating the cargo that was being delivered (Supplementary Figure S3); barcode generation with a PCR using 3′-overlapping 119-mer oligonucleotides (Supplementary Figure S4); application of targetrons as a barcode delivery system (Supplementary Figure S5); and design and test of Locus 1 and 2 spacers for CRISPR/Cas9-mediated counterselection of Ll.LtrB::B3 group II intron (Supplementary Figure S6) (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

PDF [ 5MB]

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect