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Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins
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Cite this: ACS Cent. Sci. 2021, 7, 10, 1736–1750
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https://doi.org/10.1021/acscentsci.1c00574
Published September 24, 2021

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Abstract

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The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA in vivo by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling “loopable” translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.

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Synopsis

A loopable translator enables long repetitive proteins to be efficiently synthesized in cells through a plug-and-play plasmid featuring a self-splicing intron in a permuted topology.

Introduction

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Proteins serve as the building blocks for functional, tunable materials across the tree of life. Spider silks (composed of spidroins (1−6)), connective matrices (predominantly composed of collagens (7−9)), biofilms, (10−13) and squid ring teeth (composed of SRT proteins (14−16)) are four examples of materials with distinct mechanical properties and biological functions whose properties are genetically encoded through their respective protein sequences. Nevertheless, there remain many opportunities and challenges toward the capacity to synthetically recapitulate (or improve upon) the biological processes responsible for the creation of these materials.
Recently, there have been several successes in the design of novel globular cagelike protein materials, generally based on motifs that self-assemble through symmetry. (17−20) On the other hand, natural fibrous proteins typically consist of highly repetitive low-complexity regions within polypeptides with long chain lengths and display self-assembly on many length scales, spanning from the nanometer (protein–protein interactions) to the micrometer (phase separation) and the millimeter (filamentization). Proteins of this type are generally less amenable to rational design and also are typically challenging to express.
Despite the sequence and functional diversity of extant fibrous proteins attested in nature, (1−16) some unifying features of these proteins are the presence of long, repetitive, low-complexity regions. Pioneering work by Kaplan and co-workers (1,6) demonstrated an approach to reproduce such a protein architecture in a recombinant expression vector. In their strategy, a short repetitive unit is autoligated into tandem repeats by availing of self-complementary sticky ends from two restriction enzymes. (1,6) The judicious choice of two enzymes that create identical sticky ends but recognize distinct recognition sites enables directionality and prevents the formation of inverse repeats (Figure 1A).

Figure 1

Figure 1. Approaches to generate tandem repeat proteins. (A) A plasmid encoding a monomeric unit is cut with two restriction enzymes to generate an insert. The original plasmid is cut with one of the two restriction enzymes to create self-complementary sticky ends, which allows ligation of the insert back into its original vector, creating a plasmid that encodes a tandem repeat. Upon ligation in a tail-to-head manner, the restriction site is destroyed, allowing directional cloning (inverse repeats can be removed by redigesting). This process can be repeated to generate larger 2n multimers. (B) A short segment of DNA encoding a monomeric unit is digested and circularized in vitro. The circular DNA serves as the template for rolling circle amplification and generates a mixture of concatemers with different lengths. The desired size of the repetitive sequences can be selected by gel extraction, digested, and cloned back into an expression vector. (C) In loopable translation, a plasmid is created in which a segment of DNA encoding a monomeric unit is incorporated within a permuted self-splicing Group I intron (gray blocks). The plasmid is transformed into an expression strain, and its gene is transcribed into RNA, which is circularized. The circular mRNA translates into a repetitive protein product through ribosome looping.

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More recently, work by Demirel and co-workers appropriated rolling circle amplification (RCA) (21) as an alternative strategy to generate tandem repeat proteins, an approach that has been used to express large SRT polypeptides (42 repeats; 1260 residues) in Escherichia coli (Figure 1B). (14) Nevertheless, existing technologies to generate large repetitive proteins for recombinant expression in microbial hosts suffer from a number of major limitations: (i) Highly repetitive DNA sequences suffer from genetic instability due to spontaneous recombination in microbial hosts. (ii) Large plasmids containing tandem repeat proteins can be inconvenient for routine molecular biology manipulations (e.g., PCR, sequencing, and transformation). (iii) The metabolic cost to replicate and transcribe large DNA and mRNA molecules undermines the goal of redirecting resources toward the creation of proteins. To address these limitations, we report an alternative approach based on loopable translation (Figure 1C).
In contrast to previous approaches to generate tandem repeat proteins (Figure 1A,B), the loopable translator does not require large repetitive DNA constructs; rather, it uses a permuted group I self-splicing intron to circularize an mRNA transcript, thereby allowing ribosomes to translate a region of interest many times in succession as a single polypeptide chain. Expanding on the pioneering work of Ares and co-workers (22−24) on permuted introns, we demonstrate a reporter system that can quantify the efficiency of circular translation, allowing us to optimize its performance through rational considerations and by directed evolution. Our results suggest that such a system is a promising platform for the creation of fibrous proteins with a repetitive sequence and hierarchical structure.

Results

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Creation of a Loopable Translator Coupled to a Fluorescence Reporter

Ares and co-workers demonstrated that the self-splicing group I intron within the thymidylate synthase (td) gene of T4 phage retains activity following circular permutation at the P6a stem (Figure 2A). (22−26) In this reorganized topology, the same two phosphoryl transfer reactions result in the circularization of an internal region, in contrast with the wild-type intron which catalyzes the splicing of two flanking exons. Such a circular mRNA can then be translated into a large repetitive protein. However, this remarkable activity is challenging to quantify, and indeed, tandem repeats of green fluorescent protein (GFP) are not fluorescent (possibly because of efficient nonradiative energy transfer within the polymer). In our design, superfolder GFP (sfGFP) was interrupted after its internal helix (at residue 52) with a T4 td intron (including 15 nucleotides of td exonic context flanking the intron). The exonic sequences were included to ensure proper interactions with the internal guide sequences (IGSs) of the T4 td intron (circularly permuted at P6a for circularization; Figure 2A,B). We then added a ribosome binding site (RBS) before sfGFP’s N-terminus and a TEV protease cleavage site following sfGFP’s C-terminus (Figure 2A,B) and cloned the sequence into an expression vector in which transcription was controlled by an arabinose promoter and an rrnB-T1 terminator. This plasmid (pBAD-td3′-sfGFP53-end-TEV-RBS-DB-sfGFP1–52-td5′; note that the downstream box sequence (DB) refers to a sequence reported to enhance translational efficiency (27,28)) was then cotransformed into E. coli BL21(DE3) with pRK793 (expressing a soluble form of TEV protease (TEVP)). (29,30) To abbreviate, we refer to our designed loopable translator construct as pBAD-tdTEVDB. In this system, circularized mRNA translates a GFP concatemer; however, the activity of TEVP liberates GFP monomers from the chain allowing for fluorescence to be monitored in vivo (Figure 2B).

Figure 2

Figure 2. Design of a loopable translator with a coupled fluorescence GFP reporter. (A) Sequence, secondary structure, and mechanism of the self-splicing Group I intron from the thymidylate synthase (td) gene of T4 bacteriophage. This intron catalyzes two consecutive, site-specific phosphoryl transfer reactions, which, in the natural form, results in the splicing of the two flanking exons (orange). The 5′ splice site (marked with a red arrow) is selected by base-pairing between the 5′ exonic sequence (5′ Ex) and an internal guide sequence (IGS) at the beginning of the intron (P1), while the 3′ splice site (marked with an orange arrow) is selected by a short 2 bp stem formed between the 3′ exonic sequence (3′ Ex) and the edge of the P1 loop (termed P10, shown by two gray lines). An exogenous guanosine (red) is required to initiate the reaction and becomes prepended to the 5′-terminus of the intron sequence (black). 5′* and 3′* denote the termini of the RNA molecule in its natural form. In our construct, the circular permutation in the ORF region of P6a results in two new termini (labeled 5′ and 3′), and a permuted GFP sequence (green) is incorporated between 3′* and 5′*. In this reorganized topology, intron activity will result in circularization of the internal “exonic” region. (B) Design of a GFP fluorescence reporter system for RNA circularization. A plasmid is created (pBAD-tdTEVDB) in which a permuted superfolder GFP (sfGFP) gene is incorporated within the permuted intron. The GFP is split such that the N-terminal portion (residues 1–52) is placed downstream of the C-terminal portion (residues 53–241). In between these two coding regions are inserted a TEV protease site and a ribosome binding site (RBS) along with an enhancing downstream box (DB) in frame with the GFP coding sequence. After intron folding and RNA circularization, an mRNA is formed which is competent to recruit ribosomes and generate full-length GFP. Because polymeric GFP is found to have low fluorescence, this system is coexpressed with TEV protease, which can liberate fluorescent GFP monomers from the primary chain product. (C) A negative control plasmid (pBAD-sfGFP1–52) encodes the protein product that would form in the absence of circularization (with the same promoter, origin, and selectable marker as pBAD-tdTEVDB). A positive control plasmid (pBAD-sfGFP) encodes the protein product that would form upon circularization. Importantly, it differs from a wild-type sfGFP in that it has a 10-residue long “scar sequence” in between residues 52 and 53, which result from exonic context sequences (represented as orange boxes) that were retained to ensure proper base-pairing with the IGSs. pBAD-tdTEVDB-STOP is identical to pBAD-tdTEVDB except that a stop codon is placed at the end of GFP. (D) pBAD-tdTEVDB generates a fluorescence signal that is significantly higher than the background level (P < 0.0001 by Student’s t test) but represents 4.2% of the signal of the corresponding positive control (n = 3). Fluorescence measurements were conducted in biological triplicate.

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pBAD-tdTEVDB was designed in a way that a nonfluorescent GFP fragment (residue 1–52) would be formed upon translation of a noncircularized RNA (translation was designed to terminate with a UAA stop codon that follows immediately after the end of the 5′ exonic sequence (5′ Ex); Figure 2A,B). Thus, a failure of the mRNA molecule to circularize will result in truncated GFP, which is nonfluorescent and serves as a negative control (Figure 2C). On the other hand, circularization (along with TEVP activity) will result in full-length sfGFP with a 10 amino acid scar between residues 52 and 53, arising from the natural exonic sequences of td (shown in orange in Figure 2A or as orange blocks in Figure 2B,C). This GFP is still fluorescent, and we use it as a positive control (Figure 2C). Preliminary fluorescence assays demonstrated that pBAD-tdTEVDB produces significant levels of fluorescence above baseline levels (Figure 2D, Figure S1; P < 0.0001 by Student’s t test), confirming that mRNA circularization occurs, in accordance with previous investigators’ work on this system. (22−24) Nevertheless, our fluorometric readout makes it clear that significantly less GFP (4.2%; Figure S1) is produced compared to a positive control in which proteins are expressed via standard (vectorial) translation, suggesting that either (i) RNA circularization, (ii) ribosomal initiation, or (iii) TEV activity is limiting. To test for possible toxicities associated with loopable translation, the three constructs corresponding to a negative control, positive control, and pBAD-tdTEVDB were expressed in BL21+pRK793 cells at 30 °C, and growth curves were measured (Figure S2). We found no significant difference in growth rates across these three strains.

Rational Improvement of Activity from Loopable Translator

We next sought to improve the activity of pBAD-tdTEVDB by considering a number of factors that could influence the three considerations above. Group I self-splicing introns require two cofactors: magnesium cations and a guanosine nucleoside. (31,32) Guanosine is required because the intron uses a two-step mechanism that begins with the free nucleoside cleaving the phosphodiester linkage between the 5′-exon and the intron by prepending itself to the 5′-most nucleotide of the intron, and divalent cations are required to stabilize the tertiary fold of the catalytic core. (25,31−34) We found that GFP expression from pBAD-tdTEVDB was not strongly dependent on guanosine (Figure 3A); however, fluorescence could be markedly improved by 147% (from 5.09% to 7.46% of the positive control) by increasing the concentration of MgCl2 in the growth media up to 20 mM (Figure 3B). These findings are broadly consistent with the facts that intracellular total guanosine ((p)(p)(p)GOH) concentration is quite high in E. coli (5 mM, higher than the intron’s KM (35)); however, free Mg2+ in the cytosol is relatively scarce (0.01–1 mM), and many in vitro studies of ribozymes show a strong reliance on (and benefit from) high divalent cation concentrations. (35)

Figure 3

Figure 3. High Mg and reduced temperature enhance loopable translation. (A) Bar chart showing the levels of fluorescence of several constructs (negative control (−), positive control (+), and pBAD-tdTEVDB) expressed at 37 °C with varying concentrations of guanosine supplemented to the growth media during expression assays. Additional guanosine had no significant effect on the fluorescence signal generated by pBAD-tdTEVDB. (B) Bar chart showing the levels of fluorescence of the same set of constructs expressed at 37 °C with varying concentrations of MgCl2 supplemented to the growth media during expressions assays. Higher MgCl2 concentrations had a beneficial effect; relative to 1 mM MgCl2, fluorescence levels in 20 mM MgCl2 were 1.2-fold higher (P-value = 0.01). (C) Bar chart showing the levels of fluorescence of the same set of constructs with varying MgCl2 supplemented to the growth media, but with expressions carried out at 30 °C instead of 37 °C. Lower temperature had a significantly beneficial effect on fluorescence signal (2.8-fold at 20 mM MgCl2, P-value <0.0001). Moreover, at the lower temperature, the fluorescence signal significantly benefitted from high concentrations of MgCl2 (a: 1.9-fold, P-value = 0.0006). Under these conditions, the loopable translator generated 16% of the fluorescence of the positive control. Fluorescence levels from pBAD-tdTEVDB-STOP (see Figure 2C, labeled “STOP”) were slightly higher than those from the negative control (b: 1.05-fold, P-value = 0.04), but much less than the loopable translator (c: 4.8-fold, P-value <0.0001). Fluorescence measurements were conducted in biological triplicate; statistical tests were conducted with Student’s t test.

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Strikingly, we observed an enhancement in GFP expression when cells were incubated at a lower growth temperature (30 °C), with the fluorescence levels approaching 16% of the positive control (Figure 3C). We speculate two potential reasons why this could occur: (i) TEVP is an aggregation-prone protein, and reduced temperatures could improve its solubility in the E. coli cytoplasm; and (ii) lower temperatures could shift equilibria to favor certain catalytic elements on the intron, some of which consist of only two base pairs (e.g., P10; cf. Figure 2A). (36)
As a control, we also performed a fluorescence assay with a modified construct in which a stop codon is inserted after the GFP coding sequence (pBAD-tdTEVDB-STOP, Figure 2C), such that each ribosomal initiation event on circularized mRNA would only result in a single GFP protein being synthesized (noncircularized mRNA would still result in truncated GFP). Fluorescence levels decreased markedly, down to levels only slightly higher than the baseline level associated with the negative control (Figure 3C). This result suggests that the fluorescence signal achieved by pBAD-tdTEVDB arises from ribosomes transiting circular mRNAs many times without stopping. Using the slightly higher (but still statistically significant) fluorescence signal of pBAD-tdTEVDB-STOP relative to the negative control (1.04-fold, P-value = 0.04 by Student’s t test), we can estimate the loop count (number of transits per initiation) to be ∼90 ± 55 using
where F corresponds to average fluorescence, and “loop”, “–”, and “stop” correspond to the tdTEVDB, negative control, and tdTEVDB-STOP constructs, respectively.
A number of other variations we considered did not improve the efficiency of GFP fluorescence, including an attempt to stabilize the permutation site at P6a (Figure S3, Supporting Information Text 1), flanking the TEV cleavage site with flexible linkers (GGSGGGSGG) to facilitate the access of TEV protease (Figure S4, Supporting Information Text 2), the replacement of a classic TEV protease with several mutants that have been reported to be more active (Figure S5, Supporting Information Text 3), (37−39) and the replacement of the RBS with an internal ribosome entry site (IRES) that has been reported to efficiently recruit bacterial ribosomes (Figure S6, Supporting Information Text 4). (40) We were able to detect a very low activity using an alternative circularization strategy that is based on ribozymes and RtcB ligation (Figure S7, Supporting Information Text 5), (41) though we note that this strategy was developed in mammalian cells, and not in E. coli.

Visualization of Concatemer Formation and Verifying mRNA Circularization

Because polymeric GFP (polyGFP) is not fluorescent, (23) our fluorescence assays require the activity of TEVP to generate a readable signal. In contrast, polyGFP can be visualized directly as a protein of high molecular weight via Western blot. Indeed, we found that, in the absence of the plasmid that expresses TEVP, pBAD-tdTEVDB generates a set of protein products with high molecular weights (>250 kDa) that are specific for an anti-His antibody (the GFP coding sequence contains a His-tag, Figure 4A, Figure S11). In contrast, in the presence of TEVP, pBAD-tdTEVDB generates a single band at the expected molecular weight for monomeric GFP (25 kDa), identical to a positive control in which GFP is expressed by linear translation (Figure 4A). Moreover, the introduction of a stop codon within the mRNA loop completely abrogates the high-molecular-weight features, as expected, and also significantly reduces protein expression level—showing that polyGFP synthesis is dependent on a ribosome iterating numerous times on circular mRNA. Densitometry analysis conducted on blots of biological triplicates (Figure 4B) showed that, relative to linear translation, overall protein expression from the loopable translator was down ∼3.6-fold, and protein expression from pBAD-tdTEVDB-STOP was down ∼10-fold, consistent with what we found in fluorescence assays (Figure 3C).

Figure 4

Figure 4. Verification of RNA circularization and polyGFP synthesis. (A) Anti-His Western blot image showing the protein products of pBAD-sfGFP, pBAD-tdTEVDB, pBAD-tdTEVDB-STOP, and pBAD-tdTEVDB-mCherry that were expressed with and without the pRK793 plasmid (expressing TEV protease). pBAD-tdTEVDB in the absence of TEV protease generated proteins of high molecular weight. Expression in the presence of TEV protease generates a species with the molecular weight of monomeric GFP (25 kDa). (B) Densitometry analysis showing average intensities relative to a positive control, for three biological replicates of the Western blot. (C) Construct maps of the positive control for the mCherry experiment (pBAD-sfGFP(52-DVFLGLPFNI)-mCherry), pBAD-tdTEVDB, and pBAD-tdTEVDB-mCherry. pBAD-tdTEVDB-mCherry was designed to form a larger mRNA loop compared to that of pBAD-tdTEVDB, in which GFP would be expressed only upon circularization, whereas mCherry would be expressed independently of circularization. (D) Bar chart showing the levels of fluorescence from the constructs shown in part C at 30 °C with varying concentrations of MgCl2. The positive control shows the native difference in fluorescence between GFP and mCherry and serves as a normalization factor. With pBAD-tdTEVDB-mCherry, 25% of the target mRNA achieved circularization at 20 mM Mg2+. The larger mRNA loop was detrimental for circularization, leading to lower levels of GFP fluorescence compared to those of pBAD-tdTEVDB (P = 0.0066 by Student’s t test).

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To provide independent verification that mRNA is circularized by the split-intron architecture, we designed a dual-fluorescence ratiometric construct (Figure 4C). The construct, called pBAD-tdTEVDB-mCherry, places a full-length mCherry open reading frame between the RBS and the N-terminal fragment of sfGFP. In this construct, mCherry is expressed independently of circularization while GFP, as before, requires mRNA circularization to be fully synthesized. From this construct, we found that the GFP signal was 4-fold less than that of mCherry (after correcting for background and intrinsic difference in sfGFP’s and mCherry’s fluorescence intensity, Figure 4D), suggesting that 25% of the tdTEVDB-mCherry mRNA is circularized in the steady state. We also found that the overall GFP fluorescence signal in this larger 1563-nt loop was significantly reduced (3-fold) relative to the smaller 816-nt loop created by pBAD-tdTEVDB, which is consistent with the Western blot showing 4-fold reduced polyGFP from pBAD-tdTEVDB-mCherry relative to pBAD-tdTEVDB (Figure 4A,B). Tentatively, our explanation for this observation is that forming the larger loop has a higher entropy cost to bring the two portions of the split-intron together and assemble the active ribozyme. If this difference in GFP signal can be ascribed solely to the fraction of mRNA molecules that are circularized, these data suggest that as much as ∼75% of the tdTEVDB mRNA is circularized in the steady state. Although there could be other factors involved in GFP’s translational efficiency from these two circular RNAs, Northern blot analysis of the RNA from cells harbouring pBAD-tdTEVDB found that 50% of the GFP-containing RNA was circularized (Figure S8). To summarize, evidence from Western blots, Northern blots, and our dual-fluorescence assay supports the view that the split-intron can achieve a steady-state fractional circularization level of 50–75% of 800-nt regions. We found this level suitable for our purposes, though it is noted that fractional circularization could be potentially optimized with further directed evolution of the intron.

Identifying the Minimal Context Requirements for Circularization

The design of pBAD-tdTEVDB would incorporate a 10 amino acid scar at the junction site where looping occurs. Hence, we asked what the minimal “exon context” requirements for intron activity are by “walking back” the 15 nucleotides of native exon context on each flank. Classic experiments on self-splicing introns demonstrated the essentiality of the final U·G wobble pair at the 5′ splice-site and the formation of the P1 stem between the 5′-exon and the beginning of the intron sequence (5′ IGS). (25) We successively deleted td’s 5′-exon context (GAU/GUU/UUC/UUG/GGU, encoding DVFLG) one codon at a time (Figure 5A,C and Figure S9) and discovered that, remarkably, all 5 codons of td’s 5′-exon context could be removed, resulting in a slight improvement in fluorescence activity. Inspection of the RNA molecule created following this deletion revealed that coincidentally sfGFP’s residues 50–52 are Thr-Thr-Gly, which are encoded by ACC/ACC/GGU, a sequence that conserves the three critical nucleotides (GGU) immediately before the intron (forming two strong base pairs, then a wobble base pair, Figure 5B). Moreover, like the wild-type intron, this sequence creates four total Watson–Crick base pairs along P1. We wondered if the circularization efficiency could be further improved by stabilizing P1, which we tested by mutating IGS nucleotides (U12 and A14) to a base pair with the native sfGFP nucleotides (P1* in red in Figure 5B). However, surprisingly, the mutation reduced fluorescence down to baseline levels (Figure 5C), suggesting that it abrogates circularization activity. These observations imply that the presence of several (possibly two) non-Watson–Crick base pairs in P1 is necessary for activity, rather than incidental. Collectively, these observations suggest that the minimal requirements for proper selection of the 5′ exon fragment by P1 are (i) that it must end in GGU and (ii) that the preceding four nucleotides must make two Watson–Crick (but not more) base pairs with the intron’s 5′-IGS.

Figure 5

Figure 5. Minimal context requirements for loopable translator. (A) Secondary structure of the loopable translator highlighting the 10 amino acid “scar sequence” that is incorporated into the loop because of the inclusion of 30 nt of exonic context retained from the natural td gene (15 nt from the original 5′ exon, and 15 nt from the original 3′ exon). Each codon triplet is color coded, and deletions of this context sequence are represented through their corresponding color blocks in bar charts C–E. (B) Secondary structure that is formed when all 15 nt (coding for DVFLG) of the 5′ exonic context sequence are deleted. The nucleotides that correspond to residues 50–52 of GFP (green) replace the original 5′ exon to pair with the IGS. Nucleotides marked with * (in red) represent compensatory mutations in the P1 IGS (termed the modified P1*). (C) Bar chart showing the level of fluorescence from a truncation series in which the 5′ context sequence was deleted one codon at a time. (D) Bar chart showing the level of fluorescence from a truncation series in which the 3′ context sequence was deleted one codon at a time. (E) Finding the minimal context requirements. Overall, the entire 5′ context sequence can be deleted, and all but the last 3 nucleotides of the 3′ context sequence (which form P10) can be deleted. Strengthening P10 (P10*) did not have a beneficial effect. All fluorescence measurements were conducted in biological triplicate.

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We also successively abrogated td’s 3′-exon context region one codon at a time (Figure 5A,D; Figure S9) and confirmed that only the initial three nucleotides (CUA; the ones closest to the exon and marked in magenta) are required for activity. This is consistent with previous work (36) which showed that the selection of the 3′-splice site is controlled by a combination of a tertiary structure and a short (2 bp) stem (called P10) formed between the 3′-exon (3′ Ex) and P1’s loop. A deletion of the first 3 nucleotides of the 3′-exon (CUA) abrogated all circularization activity (Figure 5D), confirming the importance of these nucleotides for splice site selection and therefore circularization.
Putting these observations together, we next sought to identify the minimal exon context requirements for intron activity, so that future use of the loopable translator system would impose minimal scar sequences at the junction site (Figure 5E). Combining the insights from these two deletion series, we found that complete removal of the 5′ td-exon context and removal of the 3′ td-exon context up to the first three nucleotides (CUA) were tolerated by the permuted td intron and, in fact, provided a small (20%) activity increase with respect to the wild-type (Figure 5E). Next, to determine if intron activity could be enhanced by strengthening P10, we modified the sequence of the initial three nucleotides from CUA to CUC, which would form a third Watson–Crick base pair with the P1 loop (P10* in blue in Figure 5B,E). The resulting construct generated a signal that was slightly lower than the construct with only CUA, suggesting that two base pairs of complementarity are indeed the optimal strength for this critical interaction.
Combining the requirement for CUA immediately after the intron and the requirement of GGU immediately before the intron, our data imply that mRNA circularization can be accomplished with a scar as small as two amino acids at the junction region.

Improving Initiation on Circular mRNA by Directed Evolution

Because 50–75% of mRNA generated by pBAD-tdTEVDB is circularized (cf. Figure 4 and Figure S8), we reasoned that initiation of ribosomes on circular mRNA could be limiting the efficiency of protein expression. In E. coli, translational initiation is mediated by several factors. The interaction between the Shine–Dalgarno sequence (i.e., RBS) and the anti-Shine–Dalgarno on 16S rRNA is a well-known determinant, and many expression vectors use “strong” RBSs to increase expression. (42−44) Additionally, ribosomal protein S1 (bS1) plays a critical—but less well understood—role in mediating initiation, (45,46) which we hypothesized could be less adept at initiating translation on circular mRNA, because S1 binds upstream of the RBS, which, for most mRNA molecules, would be proximal to the 5′ terminus. On the other hand, on circular mRNAs, RBSs would not be proximal to any terminus. To test this hypothesis, we examined whether directed evolution on the initiation region (Figure 6A) would enable the creation of a modified initiation sequence better suited for circular mRNA.

Figure 6

Figure 6. Directed evolution on the initiation sequence of loopable translator with minimum context. (A) Error-prone PCR was performed on a short 36 bp amplicon encoding the initiation region [ribosome binding site (RBS), spacer, initiator methionine, and a downstream box (DB)] of the GFP reporter gene. (B) Bar chart showing the fluorescence signals from the top-performing constructs selected from the first round of directed evolution (n = 3 biological replicates following primary screen). A construct with a point mutation (G4C) generated a signal that is significantly higher than the wild-type (pBAD-tdTEVDB) (1.9-fold; P = 0.0065). (C) Histogram showing the fluorescence signal of 372 constructs screened in the first round of directed evolution relative to the wild-type. (D) Bar chart showing the fluorescence signal from the top-performing constructs selected from the second round of directed evolution (n = 3 biological replicates following primary screen). A construct with an additional mutation (C-15A/G4C) generated a signal that was ca. 3-fold higher than the wild-type (a: P = 0.0039) and ca. 2-fold higher than the single mutant (b: P = 0.033). (E) Histogram showing the fluorescence signal of 720 constructs screened in the second round of directed evolution relative to G4C. All statistical tests were conducted using Student’s t test

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Specifically, we sought to introduce several mutations into a short 36 bp region that covers the canonical RBS, its spacer, the initiator codon, and the downstream box. We found that conventional error-prone PCR methods were ill-suited for achieving the desired mutation density in this small amplicon (1–2 mutations per 36 bp) while also maintaining low levels of bias; (47−50) hence, we developed a modified error-prone PCR method based on iterating between dilution and reamplification with error-prone touchdown PCR to suppress accumulation of incorrect products. The details of this modified method are reported elsewhere. (51)
This dilution/reamplification error-prone PCR method was used to introduce mutations into a 36 base pair region, which was then cloned back into the pBAD-tdTEVDB vector linearized with the reverse complement primers (red primers, in Figure 6A), via in-fusion cloning (see the Materials and Methods section). The library DNA was isolated and retransformed into BL21(DE3) cells harboring the pRK793 plasmid, and hundreds of colonies were selected from agar plates at random for inoculation into 96-well plates to screen for beneficial mutations by fluorescence. In the first screen, we assayed fluorescence from 372 constructs and found a bimodal distribution (Figure 6C) with many clones having a neutral phenotype (fractional performance close to 1) or with very low activity (fractional performance close to 0.2). A small number of variants had improved levels of translational efficiency, up to 1.9-fold, including a variant E12 (which contained the mutation G4C, immediately following initiator methionine; Figure 6B). Because other high performers by this screen contained mixtures of sequences upon performing sequence analysis, we focused on G4C as a starting point for a second round of diversification and screening. We used the pBAD-tdTEVDBG4C plasmid as a template for error-prone PCR to incorporate further mutations in the initiation region and cloned back into the pBAD-tdTEVDB backbone to perform a second screen. This time, we assayed 720 constructs and discovered mutants that improved translational efficiency 1.8-fold again (Figure 6E). The best performing mutant, C-15A/G4C contains an additional mutation that apparently “extends” its RBS sequence to now include a run of 8 purines; other successful clones contained other mutants at position 4. Overall, the double mutant C-15A/G4C in the initiation region is ca. 3-fold more active at producing GFP relative to our starting construct (Figure 6D). These experiments appear to support our hypothesis that ribosomal initiation rates hampered translation on circular mRNA, though specific alterations to the initiation region can mitigate this limitation. Finally, if we combine these improvements in translational initiation with lowered temperature and increased Mg2+ concentrations, the result is a ∼14-fold improvement in translational efficiency relative to the original loopable translator (cf. Figure 6D and Figure 2D, ca. 75 000 and 5260 relative fluorescence units, respectively, following background subtraction). Overall, protein expression from these optimized circular mRNAs is within a factor of 1.5 relative to the expression levels from standard linear mRNA.

Discussion and Future Direction

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Loopable translation has several traits that could make it an attractive option for the preparation of highly repetitive proteins. Because it does not require the creation of repetitive DNA sequences, the resulting genetic constructs would be expected to be more stable and less susceptible to homologous recombination. Moreover, the absence of repetitive sequences renders these plasmids amenable to many more molecular biology and synthetic biology manipulations—such as PCR, Gibson assembly, and recombineering—which are challenging (or impossible) to conduct with highly repetitive DNA sequences. These features should allow for greater interoperability; that is, it is facile for any researcher to “swap out” the GFP sequence in pBAD-tdTEVDB with an arbitrary sequence via a single Gibson assembly. All of these features additionally make this system much more amenable to creating combinatorial libraries and performing directed evolution, as we have demonstrated. Due to the universal nature of the autocatalytic intron splicing reaction (requiring only magnesium and guanosine as cofactors), we expect that circularization should be functional in a range of microbial hosts. Finally, the repeat number (loop count) from such a system could be very high—as the data with the pBAD-tdTEVDB-STOP construct (Figure 3C) would suggest—possibly only limited by the presumably rare occurrence of spontaneous frame-shifting (52) or possibly nonspecific peptide release from the ribosome.
Initially, our study focused on coupling mRNA circularization to a fluorescence-based reporter (sfGFP), so expression from circular mRNA could be quantified under a range of conditions and for a variety of constructs. This reporter proved to be valuable in identifying the importance of a high concentration of magnesium (20 mM) and lower growth temperature (30 °C) for a high level of expression, likely because both factors help folding and stability of the intron, which consists of several “delicate” components, notably P10 which specifies the 3′ splice site in the catalytic machinery with only 2 base pairs. The benefit of these factors is supported by the higher RNA circularization levels evidenced in our Northern blot compared to previous reports (cf. Figure S8 and refs (22−24)). Although expression levels from this system were initially low, we hypothesized that improved translational efficiency could be achieved via modification of the initiation region. The finding that we were able to enhance translational efficiencies nearly 3-fold upon screening a relatively small number of clones could suggest that greater improvements could be achieved with further rounds of directed evolution, an effort that we are currently undertaking.
The next major direction for this line of research is to deploy the loopable translator to create long repetitive fibrous proteins that could potentially have enhanced material properties (see the Materials and Methods section). In preliminary experiments, we were able to clone sequences corresponding to the major ampullate spidroin protein 1 (MaSp1) into the loopable translator motif and attempted to express high-molecular-weight spidroins (Figure 7A). Surprisingly, we detected that nonlooped MaSp1 proteins could generate species of high molecular weight (>250 kDa), while looped MaSp1 proteins resulted in very low expression levels overall (Figure 7B). One possible explanation for these findings is that the spidroin sequence that we used in this study is very hydrophobic and aggregation-prone, (53−56) meaning that even proteins of low nominal molecular weights might form aggregates relatively easily, while higher-molecular-weight spidroins (such as those that would be expected to form from the loopable translator) might form insoluble entities that cannot be stably expressed in the E. coli cytosol and instead engage various forms of cellular stress–response which terminate their translation and initiate their degradation.

Figure 7

Figure 7. Applying pBAD-tdTEVDB to producing spider silk spidroin. (A) Illustration of the nonlooped and looped constructs containing repetitive units of dragline silk. A repetitive unit with 36 amino acids was chosen as a monomeric unit. Six constructs were generated, comprising 8, 16, or 24 tandem repeats of a repetitive protein unit derived from major ampullate spidroin protein 1 (MaSp1), cloned into either a standard expression vector or the loopable translator. (B) Anti-His Western blot image demonstrating the protein products of the six constructs. Apparent low molecular weights from the loopable translator could be due to high insolubility and/or instability of the resulting protein products.

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Future studies will therefore have to balance the potential benefits of long fibrous proteins with the requirement of maintaining solubility and stability within the cytosol prior to downstream material processing. This might be possible by incorporating solubilizing domains, which naturally occur at the N- and C-terminus of spidroin proteins and play important roles in maintaining these proteins in a soluble micelle-like state before they are concentrated and pulled into fibers in a spider’s spinerets. (57−59) Cloned into our loopable translator motif, the solubilizing end domains will be expressed many times along with the fibrous monomeric unit and would potentially allow the expression of long repetitive fibrous protein materials with desired properties. It may also be that the loopable translator would be better suited at translating very long proteins that consist of domains which are initially soluble and then fold-switch into an aggregating form, seeing as our results have demonstrated the viability of expressing soluble domains as long concatemers with relative ease (cf. Figure 4). Curli proteins such as E. coli CsgA are potentially attractive in this regard as they have been extensively engineered (10,11,60,61) and are translated in a primary form that maintains solubility in the E. coli periplasm prior to amyloid growth nucleated following secretion. (62,63) Looped extracellular matrix (ECM) proteins such as fibronectin (64) and collagen (65) are other potential targets for applications as tissue engineering scaffolds, (66) where the loopable translator system would provide the benefits of facile genetic tuning and incorporation of signaling protein modules. Hence, we are optimistic for the future outlook of protein materials research efforts building off the tools that we have herein described.

Materials and Methods

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Cloning pBAD-tdTEVDB Plasmid Construct and Controls

To create the negative control plasmid (pBAD-sfGFP1-52), a plasmid encoding the full-length superfolder GFP (pBAD-sfGFP) was used as a template for PCR and amplified with primers Delete53-f and Delete53-r (Table S1 and Figure S10) using the Q5 DNA polymerase (NEB) according to the manufacturer’s protocol. The construct was designed in a way that the truncated GFP is under the control of the same arabinose-inducible promoter and terminator. The PCR product was assessed by 0.8% agarose (0.5× TBE) gel electrophoresis for the correct molecular weight, DpnI (NEB) digested (10 U were added to the PCR reaction and incubated at 37 °C for 30 min and then 80 °C for 20 min to inactivate), column purified using the DNA clean-and-concentrate kit (Zymo) according to the manufacturer’s protocol, and quantified using a Nanodrop OneC (Thermo). The DNA was ligated using the QuickChange strategy by directly transforming it into chemically competent 10-beta cells (NEB) without in vitro ligation. PCR products that are described as being ligated by QuickChange mean that the primers were designed to encode ∼15 nucleotides of homology at the 5′ and 3′ termini, enabling ligation in vivo by 10-beta cells’ endogenous recombinases (see Table S1 for details). Next, 1 μL of purified DNA was combined with 25 μL of competent cells, incubated on ice for 25 min, subject to a heat pulse at 42 °C in a water bath for 40 s, and then returned to ice for 2 min. Next, cells were recovered by inoculation into 1 mL of SOC media and incubated at 37 °C for 1 h with agitation (700 rpm in a thermomixer), and then, 100 μL of the transformant was spread out on selective plates consisting of LB agar supplemented with 15 μg/mL tetracycline using coli rollers. After ∼16 h of incubation at 37 °C, colonies were selected and inoculated into 5 mL of LB supplemented with 15 μg/mL tetracycline and incubated overnight (∼16 h) at 37 °C with agitation (220 rpm). Cells from the saturated overnight cultures were collected by centrifugation (3200g for 15 min at 4 °C), and plasmid DNA was isolated using the ZR plasmid miniprep kit (Zymo) according to the manufacturer’s protocol. In this (and in all further molecular cloning manipulations), we would typically isolate plasmid DNA from 4–6 colonies and subject it to sequencing analysis by Sanger sequencing (GeneWiz, following GeneWiz’s specifications). A single clone with the correct DNA sequence would then be used for further steps.
The positive control was created through a QuickChange approach to introduce the two td-exon context sequences (encoding the decapeptide DVFLGLPFNI) at the junction between residues 52 and 53. PCR was conducted on pBAD-sfGFP as a template and using the primers TD_Context_Insert-f and TD_Context_Insert-r (Table S1 and Figure S10). The PCR product was purified and transformed as described above. This construct creates a replica of the GFP molecule that would be created by the loopable translator.
A geneblock for the prototype loopable translator (td-FP-G-td) was ordered from IDT and cloned after the araBAD promoter in pBAD through Gibson assembly. A linearized pBAD vector was generated by PCR using pBAD-sfGFP as a template and oligos clone-pBADGFP-f and clone-pBADGFP-r as primers. The vector fragment was assessed by 0.8% agarose (0.5× TBE) gel electrophoresis for the correct molecular weight, DpnI (NEB) digested (10 U was added to the PCR reaction and incubated at 37 °C for 30 min and then 80 °C for 20 min to inactivate), and column purified (Zymo). The prototype geneblock was designed to have the permuted sfGFP (residues 53–241 and then 1–52) flanked by the permuted td intron of T4 bacteriophage (P6a–P9.2, P1–P6a). The td-FP-G-td geneblock has a His tag at the end of GFP residue 241, a stop codon at the end of the His tag, and a ribosome binding site (RBS) in front of GFP residue 1. The geneblock and the linearized vector were ligated using HiFi 2x× Gibson Master Mix (NEB) according to the manufacturer’s protocol and transformed into chemically competent 10 beta cells as described above.
Because polyGFP is not fluorescent, (23,24) we modified this system in a few more ways. A TEV cleavage site was installed after the His-tag, and the stop codon was removed using PCR with primers TEV-f and TEV-r via QuikChange. We also installed the downstream box sequence (27,28) after the start codon using PCR with primers InsertDB-F and InsertDB-R via QuikChange (Table S1 and Figure S10). (23,24) Following this sequence of alterations, the resulting construct corresponds to the pBAD-tdTEVDB drawn in Figure 2C and interrogated in Figure 3. The full nucleotide sequence of this construct is given in Figure S11.
To create pBAD-tdTEVDB-STOP, we reintroduced the TAA stop codon between GFP and the TEV site by PCR with primers TEV_DB_STOP_F and TEV_DB_STOP_R via blunt-end ligation (Table S1 and Figure S10).

Fluorescence Assays

First, chemically competent BL21(DE3) (NEB) cells were transformed with pRK793 (encoding TEV protease), (30) a gift from the laboratory of Doug Barrick (JHU Biophysics department). 1 μL of pRK793 plasmid DNA and 25 μL of competent BL21(DE3) cells were mixed together in a microfuge tube by aspiration, incubated on ice for 25 min, subject to heat pulse at 42 °C in a water bath for 40 s, and then returned to ice for 2 min. Next, cells were recovered by inoculation into 1 mL of SOC media and incubated at 37 °C for 1 h with agitation (700 rpm in a thermomixer), and then, 100 μL of the transformant was spread out on selective plates consisting of LB agar supplemented with 100 μg/mL ampicillin using coli rollers. After ∼16 h of incubation at 37 °C, one colony was inoculated into 5 mL of LB supplemented with 100 μg/mL ampicillin and incubated overnight (∼16 h) at 37 °C with agitation (220 rpm). The overnight culture was inoculated into 1 L of LB with 100 μg/mL ampicillin in a 2 L baffled sterile flask to a starting OD600 of 0.02–0.04 to make homebrew BL21(DE3)+pRK793 competent cells. The BL21(DE3)+pRK793 day culture was incubated at 37 °C with agitation (220 rpm) until the OD reached 0.4–0.6. When the desired OD was achieved, the culture was transferred into 500 mL centrifuge bottles and incubated on ice for 20 min (after this step, the culture was handled either on ice or in a cold room). During the incubation, three buffers (200 mL of 100 mM MgCl2, 200 mL of 100 mM CaCl2, and 60 mL of 85 mM CaCl2, 15% glycerol) were prepared and prechilled on ice. The ice-incubated culture was centrifuged at 3000g for 15 min at 4 °C. The supernatant was decanted off, and cell pellets in each 500 mL bottle were resuspended in 100 mL of cold 100 mM MgCl2. The resuspension was centrifuged at 2000g for 15 min at 4 °C. The supernatant was decanted off, and cell pellets in each 500 mL bottle were resuspended in 100 mL of cold 100 mM CaCl2. The resuspension was incubated on ice for 20–40 min and centrifuged at 2000g for 15 min at 4 °C. The supernatant was decanted off, and cell pellets in each 500 mL bottle were resuspended in 25 mL of cold 85 mM CaCl2 and 15% glycerol, transferred to a 50 mL falcon tube, and spun at 1500g for 15 min at 4 °C. The supernatant was decanted off, and cell pellets in the 50 mL tube were resuspended in 4 mL of cold 85 mM CaCl2 and 15% glycerol. The 4 mL of competent BL21(DE3)+pRK793 cells was aliquoted into 40 microfuge tubes, containing 105 μL apiece, which were flash frozen in liquid nitrogen and stored at −80 °C until further use.
To begin a fluorescence assay, an aliquot of BL21(DE3)+pRK793 was thawed on ice. 1 μL of pBAD plasmid DNA and 25 μL of competent BL21(DE3)+pRK793 cells were mixed together in a microfuge tube by aspiration, incubated on ice for 25 min, subject to a heat pulse at 42 °C in a water bath for 40 s, and then returned to ice for 2 min. Next, cells were recovered by inoculation into 1 mL of SOC media and incubated at 37 °C for 1 h with agitation (700 rpm in a thermomixer), and then, 40 μL of the transformant was spread out on selective plates consisting of LB agar supplemented with 100 μg/mL ampicillin and 15 μg/mL tetracycline using coli rollers. After ∼16 h of incubation at 37 °C, (typically) three separate colonies were inoculated into 3× 200 μL LB supplemented with 100 μg/mL ampicillin and 15 μg/mL tetracycline in a clear-bottom 96-well plate (Costar). After inoculation, the plate was sealed with parafilm and incubated overnight (∼16 h) at 37 °C with agitation (220 rpm), and these created the biological triplicates used for fluorescence measurements. For repeat measurements, separate colonies would be selected from the same agar plate, which were stored at 4 °C and would be reused for at most 1 week.
The final OD600 values were measured for the overnight cultures using a plate reader instrument (SpectraMax iD3 from Molecular Devices) and were used to subculture down to a starting OD600 of 0.05 in 200 μL of induction media (LB supplemented with 15 μg/mL tetracycline, 100 μg/mL ampicillin, 0.1 mM IPTG, and 0.2% arabinose) in a clear-bottom 96-well plate. 96-well plates containing biological triplicates for each of the conditions considered would be loaded into an iD3 microplate reader preincubated at 37 °C. The plate reader recorded both growth (OD600 by absorbance) as well as GFP fluorescence (excitation at 488 nm, emission at 535 nm, PMT gain at 500 V with an integration time of 20 ms from 5 mm from the plate) every 10 min over 16 h. In between measurements, the plate reader agitated the plate with orbital mixing (high; 577 rpm).
In later experiments, plate reader measurements would be conducted at 30 °C (instead of 37 °C). Additionally, induction media was additionally supplemented with various concentrations of guanosine (Sigma) or MgCl2.
To analyze the data, the absorbance graphs of the cultures were first examined to see if any data points needed to be removed due to absence of growth. Based on early experiments, it was found that the fluorescence time point after 50 000 s of growth was representative of maximal expression levels before cell death, and hence, this time point was used throughout this study. Fluorescence values at 50 000 s were compiled in Graphpad Prism 9, which was used to generate the bar charts shown in Figures 2−6. Statistical analysis was conducted using Student’s t test (assuming normally distributed populations with equal variances), as implemented in Prism 9.

Protein Expression and Western Blot

1 μL of each pBAD plasmid DNA was mixed together with either 25 μL of competent BL21(DE3) cells or 25 μL of competent BL21(DE3)+pRK793 cells, respectively, in a microfuge tube by aspiration, incubated on ice for 25 min, subject to a heat pulse at 42 °C in a water bath for 40 s, and then returned to ice for 2 min. Next, the cells were recovered by inoculation into 1 mL of SOC media and incubated at 37 °C for 1 h with agitation (700 rpm in a thermomixer), and 40 μL of the transformant was spread out on a corresponding selective plate using coli rollers; BL21(DE3) transformants were spread on a selective plate consisting of LB agar supplemented with 15 μg/mL tetracycline only, and BL21(DE3)+pRK793 transformants were spread on a selective plate consisting of LB agar supplemented with 100 μg/mL ampicillin and 15 μg/mL tetracycline. One colony from each plate was inoculated into 5 mL of LB supplemented with the corresponding antibiotics in a 14 mL sterile round-bottom tube (ThermoFisher) and grown overnight (∼16 h). These overnight cultures were subcultured down to a starting OD600 of 0.05 in 50 mL of LB media supplemented with corresponding antibiotics, inducers (0.2% arabinose and 0.1 mM IPTG), and 20 mM Mg2+ in sterile 250 mL Erlenmeyer flasks. The day cultures were grown for ∼4 h at 30 °C with agitation (220 rpm) to final ODs of 1.0 to 1.2, aliquoted into 1.5 mL microfuge tubes by 1 mL, spun down at 3000g for 15 min at 4 °C, and stored at −20 °C until future use.
For lysis, the cell pellets were thawed on ice for 15 min and resuspended in 1 mL of 1× PBS. 30 μL of each resuspension was transferred to a microfuge tube, mixed with 7.5 μL of 5× Tris-glycine-SDS loading buffer via vortexing, heated in a 90 °C water bath for 5 min, and incubated on ice for 2 min. 30 μL of each lysate was loaded onto precast Novex WedgeWell 8–16% Tris-glycine Mini protein gels (ThermoFisher Scientific) with 3 μL of prestained PAGE ruler as the ladder (ThermoFisher Scientific; 26619) and was separated by electrophoresis at 140 V for approximately 1 h, using 1× Tris-glycine-SDS electrophoresis running buffer (BioRad). The resulting gel was incubated for approximately 2–3 min in 0.8× Tris-glycine buffer (BioRad) and 20% (v/v) methanol. The gel was trimmed to remove the wells and foot, and electroblotting was performed using an iBlot 2 gel transfer device (ThermoFisher) and its matching transfer packet (Invitrogen; iBlot 2 Transfer Stacks, PVDF, regular size), according to the manufacturer’s protocol (7 min; 20 V). After electroblotting, the PVDF membrane was incubated in 15 mL of 5% (w/v) nonfat milk (Nestle—Carnation, instant nonfat dry milk)–TBST solution for approximately 1 h with rocking to block the membrane. 5% nonfat milk–TBST solution was made by combining 1× TBS (Quality Biological; pH 7.4) with evaporated milk and Tween 20 to a final concentration of 0.1% (v/v). The blocked membrane was incubated ∼16 h in 8 mL of diluted primary anti-His antibody (mouse; Invitrogen) solution at 4 °C with rocking; the diluted primary antibody was made by diluting the antibody by 1:1000 in 5% (w/v) nonfat milk–TBST. The membrane was rinsed in 1× TBST 3 times for 10 min each and incubated in 8 mL of diluted secondary antimouse-HRP antibody (goat; Invitrogen) solution at room temperature for 40 min with rocking; the diluted secondary antibody was made by diluting the antibody 1:10 000 in 5% (w/v) nonfat milk–TBST. The incubated membrane was rinsed in 1× TBST 3 times for 10 min each, incubated for 1 min in 800 μL of chemiluminescence reagents (Signal West Femto Maximum Sensitive Substrate; ThermoFisher Scientific) that were mixed in a 1:1 ratio, and then, images were acquired using a ChemiDoc Touch imaging system (BioRad).

Making Constructs for a Dual-Fluorescence Ratiometry Assay

A pRK5 plasmid containing a gene that encodes mCherry was obtained as a gift from the laboratory of TaekJip Ha (JHU Biophysics department). Using this plasmid as a template, two new constructs were made by PCR via Gibson assembly: a new positive control with mCherry (pBAD-sfGFP52-DVFLGLPFNI-P-mCherry) and pBAD-tdTEVDB with mCherry (pBAD-td-FP-TEVDB-mCherry-TEV-G-td; denoted as pBAD-tdTEVDB-mCherry) (Figure 4C). For the new positive control, a linearized vector was made by PCR using the original positive control (pBAD-sf-GF_DVFLGLPFNI_P) as the template and primers, PosCTRL_BB_F and tRBSdX_BB_R (Figure S10 and Table S1). The vector was then assessed by 0.8% agarose (0.5× TBE) gel electrophoresis and purified as described above. The mCherry insert was made by PCR using primers, mCherry-tRBSdX-F and mCherry-posCTRL_R, assessed by 0.8% agarose (0.5× TBE) gel electrophoresis, and purified as described above. The linearized vector and the inset were ligated using HiFi 2× Gibson Master Mix (NEB) according to the manufacturer’s protocol and transformed into chemically competent 10 beta cells as described above.
For mCherry-containing pBAD-tdTEVDB, pBAD-tdTEVDB was linearized by PCR using primers, TEV-DB-BB-F and TEV-DB-BB-R (Figure S10 and Table S1), assessed by 0.8% agarose (0.5× TBE) gel electrophoresis, and purified as described above. The mCherry insert was made by PCR using primers, mCherry-TEV-DB-F and mCherry-R (Figure S10 and Table S1), assessed by 0.8% agarose (0.5× TBE) gel electrophoresis, and purified as described above. The linearized vector and the inset were ligated using HiFi 2× Gibson Master Mix (NEB) according to the manufacturer’s protocol and transformed into chemically competent 10 beta cells as described above. The two new constructs were sequence verified, before being transformed into BL21(DE3)+pRK793 cells and assessed using the fluorescence assay method described below in the Dual-Fluorescence Ratiometry Assay section.

Dual-Fluorescence Ratiometry Assay

1 μL of a set of pBAD plasmids (negative control, pBAD-sfGF52-DVFLGLPFNI-P-mCherry, pBAD-tdTEVDB, and pBAD-tdTEVDB-mCherry) was transformed into BL21+pRK793 as described above. 40 μL of the transformant was spread out on selective plates consisting of LB agar supplemented with 100 μg/mL ampicillin and 15 μg/mL tetracycline using coli rollers. After ∼16 h of incubation at 37 °C, three separate colonies were inoculated into 3× 200 μL of LB supplemented with 100 μg/mL ampicillin and 15 μg/mL tetracycline in a clear-bottom 96-well plate (Costar). After inoculation, the plate was sealed with parafilm and incubated overnight (∼16 h) at 37 °C with agitation (220 rpm), and these created the biological triplicates used for fluorescence measurements. The final OD600 values were measured for the overnight cultures using a plate reader instrument (SpectraMax iD3 from Molecular Devices) and were used to subculture down to a starting OD600 of 0.05 in 200 μL of induction media (LB supplemented with 15 μg/mL tetracycline, 100 μg/mL ampicillin, 0.1 mM IPTG, 0.2% arabinose, and 20 mM Mg2+) in a clear-bottom 96-well plate. 96-well plates containing biological triplicates were loaded into an iD3 microplate reader preincubated at 30 °C and measured for both growth (OD600 by absorbance) and GFP and mCherry fluorescence (GFP, excitation at 488 nm, emission at 535 nm; mCherry, excitation at 588 nm, emission at 650 nm) every 10 min over 16 h. The rest of the setting, including PMT and the speed of orbital mixing, stayed the same.
To obtain the ratio of the native fluorescence of GFP and mCherry, the corresponding backgrounds (GFP and mCherry signals from the negative control) were subtracted from the fluorescence signals (GFP and mCherry) generated from the positive control (pBAD-sfGF52-DVFLGLPFNI-P-mCherry). For the positive control, GFP generated signals that were 54% of the level of mCherry (Figure 4D). This served as a normalization factor. For pBAD-tdTEVDB-mCherry, the corresponding backgrounds were subtracted from each of the fluorescence signals. The background-subtracted GFP signals were divided by the background-subtracted mCherry signals and then divided by the normalization factor (0.54) to generate the normalized GFP/mCherry ratio, indicating circularization efficiency.

Library Generation

To improve ribosomal initiation on the circular mRNA, error-prone PCR (epPCR) was performed on the initiation region of pBAD-tdTEVDB plasmid to install 1 or 2 point mutations on the target region. The starting point for these mutations was the pBAD-tdTEVDB variant with the trimmed-down context regions (DVFLG deleted from the 5′-exon context and PFNI deleted from the 3′-exon context, see Figure 5). The commercial GeneMorph II Random Mutagenesis kit (Agilent) was used to perform epPCR, but using a modified protocol that involved performing several iterations of dilution and reamplification with a touchdown PCR protocol (see ref (51) for details). The forward and reverse primers SD_lib_insert_F/R (Table S1 and Figure S10) were designed to span the initiation sequence. In the first round of epPCR, the manufacturer’s protocol was adopted except that template plasmid was first diluted 109-fold, and only 1 attagram (ag; 1 ag = 10–18) of plasmid DNA was used as a template in the PCR. The first epPCR product (primary amplicon) was size-verified by 1.2% agarose gel and diluted 1000-fold using Millipore water, and 0.5 μL of the diluent (ca. 2 pg) was used to seed a reamplification epPCR in a 25 μL scale that otherwise had the same primer, polymerase, and dNTP concentrations. Unlike the first epPCR, the reamplification PCR used a touchdown protocol in which the annealing temperature started at 65 °C in the first cycle and decreased by 0.5 °C in each cycle. Under these optimal reaction conditions found, the reamplification was repeated a total of nine times, in each case diluting the product by 1000-fold and using 0.5 μL of the diluent to seed the next PCR. After the final reamplification, the PCR products were subjected to a digest with DpnI and column-purified. Separately, backbone fragments were amplified using the primers SD_lib_BB_F/R (Table S1 and Figure S10) with a high-fidelity DNA polymerase (Q5 polymerase, NEB) that were designed to have homology arms with the flanks of the mutagenized insert. The insert was then cloned into the backbone via in-fusion cloning (TaKaRa Bio Inc.), and the ligated products were transformed into chemically competent 10 beta cells as described previously. The transformant was directly inoculated into liquid culture, from which plasmid DNA was purified via midi-prep to generate a plasmid library.

Directed Evolution

1 μL of the plasmid library was transformed into 25 μL of BL21(DE3)+pRK793 competent cells as previously described, and 40 μL of the transformant was spread over selective plates of LB agar supplemented with 15 μg/mL tetracycline and 100 μg/mL ampicillin using coli rollers: 4 separate plates were prepared for the first round of directed evolution, and 8 plates were prepared for the second round of directed evolution. As a reference sample, pBAD-tdTEVDB was also transformed into 25 μL of BL21(DE3)+pRK793 competent cells as previously described. After ∼16 h of incubation at 37 °C, three colonies of pBAD-tdTEVDB were manually picked from the plate using sterile pipet tips and were inoculated into the first three wells (A1–A3) of each of the four Nunc 96-Well Polypropylene DeepWell plates (2 mL well capacity; Fisher Scientific); each well contained 1.2 mL of LB media supplemented with 15 μg/mL tetracycline and 100 μg/mL ampicillin. The colonies on the plasmid library plates were automatically picked by sterilized needles of the RapidPick Colony Picking system (Wagner Life Science) through its corresponding software (Picker 5.0.1); 372 colonies were picked for the first round of directed evolution, and 720 colonies were picked for the second round. The picked colonies were inoculated into the identical selective media in each of the remaining wells of the 96 DeepWell plates. After inoculation, the 96 DeepWell plates were covered with breathable sheets [Breathe-EASIER 6” × 3.25” (cat. no. BERM-2000); Diversified BioTech] and incubated at 37 °C overnight with agitation (220 rpm). After ∼16 h of incubation, 5 μL of each overnight culture was inoculated into 200 μL of LB supplemented 15 μg/mL tetracycline, 100 μg/mL ampicillin, 0.1 mM IPTG, 0.2% arabinose, and 20 mM MgCl2 on black flat-bottom 96-well plates [4 plates; Caplugs Evergreen Labware Products (cat. no. 290-8195-Z1F)] using a 20 μL multichannel pipet (Eppendorf). The inoculated cultures in the black 96-well plates were again covered with breathable sheets and incubated at 30 °C with agitation (300 rpm) for 13 h (the 13 h time point was selected for measuring fluorescent signals based on the signal curve patterns of constructs observed up to that point). At the 13 h time point, all plates were taken out from the shaker and directly screened for fluorescent signals (excitation at 488 nm; emission at 535 nm; without lid) by a Spark multimode plate reader (Tecan). Top-performing constructs were chosen and directly inoculated into LB supplemented with 15 μg/mL tetracycline and 100 μg/mL ampicillin, from which constructs were purified via mini-prep and sent off for Sanger sequencing. Once sequence-verified, 1 μL of each of the purified constructs was rephenotyped by transformation into 25 μL BL21(DE3)+pRK793 competent cells and used to set up a fluorescence assay in biological triplicates.

Safety Statement

No unexpected or unusually high safety hazards were encountered.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00574.

  • Additional methods, data, and figures including fluorescence levels, performances, OD600 values, max growth rate, structures, illustrations, Northern blot image, densitometry results, flow chart, and sequences (PDF)

Accession Codes

Plasmid Availability: pBAD-tdTEVDB_C-15A/G4C (the reporter plasmid following two rounds of directed evolution) is available on AddGene.

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  • Authors
  • Author Contributions

    Conceptualization was performed by S.D.F. Methodology was provided by S.O.L. and S.D.F. Investigation was performed by S.O.L., Q.X., and S.D.F. Visualization was performed by S.O.L., Q.X., and S.D.F. S.D.F. supervised the work. S.O.L. and S.D.F wrote the manuscript.

  • Notes
    The authors declare the following competing financial interest(s): The loopable translator is currently under review by JHTV for a pending patent.

Acknowledgments

Click to copy section linkSection link copied!

We acknowledge HFSP (RGY0074/2019) for funding, as well as start-up funding from Johns Hopkins.

References

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This article references 66 other publications.

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    Bale, Jacob B.; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O.; Gonen, Tamir; King, Neil P.; Baker, David
    Science (Washington, DC, United States) (2016), 353 (6297), 389-394CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Nature provides many examples of self- and co-assembling protein-based mol. machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochem. reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, the authors report the computational design and exptl. characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with mol. wts. (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nm in diam.) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallog. show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of mol. cargo through charge complementarity. The ability to design megadalton-scale materials with at.-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based mol. machines.
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  18. 18
    King, N. P.; Jacobitz, A. W.; Sawaya, M. R.; Goldschmidt, L.; Yeates, T. O. Structure and Folding of a Designed Knotted Protein. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (48), 20732– 20737,  DOI: 10.1073/pnas.1007602107
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    18
    Structure and folding of a designed knotted protein
    King, Neil P.; Jacobitz, Alex W.; Sawaya, Michael R.; Goldschmidt, Lukasz; Yeates, Todd O.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (48), 20732-20737, S20732/1-S20732/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A very small no. of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophys. characterization and x-ray crystal structure detn. show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approx. 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic expts. suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy - connecting two monomers of an intertwined homodimer into a single protein chain - supports a model for evolution of knotted structures via gene duplication.
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  19. 19
    Hershewe, J. M.; Wiseman, W. D.; Kath, J. E.; Buck, C. C.; Gupta, M. K.; Dennis, P. B.; Naik, R. R.; Jewett, M. C. Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12. ACS Synth. Biol. 2020, 9 (12), 3388– 3399,  DOI: 10.1021/acssynbio.0c00442
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    19
    Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12
    Hershewe, Jasmine M.; Wiseman, William D.; Kath, James E.; Buck, Chelsea C.; Gupta, Maneesh K.; Dennis, Patrick B.; Naik, Rajesh R.; Jewett, Michael C.
    ACS Synthetic Biology (2020), 9 (12), 3388-3399CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramol. networks of nanoconfined β-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concn. on S12 soly., secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ~ 100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and β-sheet content by PEGylation. This work presents new insights into the biochem. of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.
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  20. 20
    Votteler, J.; Ogohara, C.; Yi, S.; Hsia, Y.; Nattermann, U.; Belnap, D. M.; King, N. P.; Sundquist, W. I. Designed Proteins Induce the Formation of Nanocage-Containing Extracellular Vesicles. Nature 2016, 540 (7632), 292– 295,  DOI: 10.1038/nature20607
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    20
    Designed proteins induce the formation of nanocage-containing extracellular vesicles
    Votteler, Jorg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.
    Nature (London, United Kingdom) (2016), 540 (7632), 292-295CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Complex biol. processes are often performed by self-organizing nanostructures comprising multiple classes of macromols., such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biol. materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochem. analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (∼100 nm) vesicles contg. multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biol. materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer mols. between cells.
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  21. 21
    Jung, H.; Pena-Francesch, A.; Saadat, A.; Sebastian, A.; Kim, D. H.; Hamilton, R. F.; Albert, I.; Allen, B. D.; Demirel, M. C. Molecular Tandem Repeat Strategy for Elucidating Mechanical Properties of High-Strength Proteins. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (23), 6478– 6483,  DOI: 10.1073/pnas.1521645113
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    21
    Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins
    Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (23), 6478-6483CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mech. properties remains elusive. We propose a new approach for the design and prodn. of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and cryst. domains. We produced segmented polypeptides with varying repeat no., while keeping the lengths and compns. of the amorphous and cryst. regions fixed. We showed that mech. properties of these synthetic proteins could be tuned by modulating their mol. wts. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the no. of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.
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  22. 22
    Ford, E.; Ares, M., Jr Synthesis of Circular RNA in Bacteria and Yeast Using RNA Cyclase Ribozymes Derived from a Group I Intron of Phage T4. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (8), 3117– 3121,  DOI: 10.1073/pnas.91.8.3117
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    22
    Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4
    Ford, Ethan; Ares, Manuel, Jr.
    Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (8), 3117-21CODEN: PNASA6; ISSN:0027-8424.
    Studies on the function of circular RAN and RNA topol. in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). The authors show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, contg. 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topol., efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.
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  23. 23
    Perriman, R.; Ares, M. Circular MRNA Can Direct Translation of Extremely Long Repeating- Sequence Proteins in Vivo. RNA 1998, 4 (9), 1047– 1054,  DOI: 10.1017/S135583829898061X
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    23
    Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo
    Perriman, Rhonda; Ares, Manuel, Jr.
    RNA (1998), 4 (9), 1047-1054CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant prodn. of such proteins for structural and engineering studies, we have produced circular mRNAs with infinite open reading frames. We show that a circular mRNA contg. a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Anal. of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technol. provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.
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    Perriman, R. Circular MRNA Encoding for Monomeric and Polymeric Green Fluorescent Protein. Methods Mol. Biol. 2002, 183, 69– 85,  DOI: 10.1385/1-59259-280-5:069
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    Circular mRNA encoding for monomeric and polymeric green fluorescent protein
    Perriman, Rhonda
    Methods in Molecular Biology (Totowa, NJ, United States) (2002), 183 (Green Fluorescent Protein), 69-85CODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)
    To facilitate recombinant prodn. of very long repeating proteins (e.g., silks, mollusk shell framework, etc.), the author has developed a method for producing mRNAs on circular RNA templates. This circularization process is derived from a rearranged group I intron, from which circular RNA is produced through the splicing activity of autocatalytic group I RNA elements. Because the only cofactors required for spicing of the group I intron are magnesium and guanosine, the process can take place in a variety of organisms, making it amenable to a wide variety of protein expression systems. This chapter details the design and construction of circular RNAs contg. the open reading frame encoding for green fluorescent protein (GFP). Included on the circular GFP mRNA constructs are translation initiation sequences designed to recruit either prokaryotic or eukaryotic ribosomes. The mRNAs produce extremely long protein chains of polyGFP, demonstrating that both prokaryotic and eukaryotic ribosomes can internally initiate and repeatedly transit a circular mRNA. Protocols are presented for constructing plasmids for prodn. of circular mRNA, for testing them, and for expressing the protein in Escherichia coli and in rabbit reticulocyte lysates.
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  25. 25
    Shub, D. A.; Gott, J. M.; Xu, M. Q.; Lang, B. F.; Michel, F.; Tomaschewski, J.; Pedersen-Lane, J.; Belfort, M. Structural Conservation among Three Homologous Introns of Bacteriophage T4 and the Group I Introns of Eukaryotes. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (4), 1151– 1155,  DOI: 10.1073/pnas.85.4.1151
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    25
    Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes
    Shub, David A.; Gott, Jonatha M.; Xu, Ming Qun; Lang, B. Franz; Michel, Francois; Tomaschewski, Joerg; Pedersen-Lane, Joan; Belfort, Marlene
    Proceedings of the National Academy of Sciences of the United States of America (1988), 85 (4), 1151-5CODEN: PNASA6; ISSN:0027-8424.
    Three group I introns of phage T4 were compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the 2 newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, contg. phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the 3 introns. All 3 introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all 3 ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.
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    Umekage, S.; Uehara, T.; Fujita, Y.; Suzuki, H.; Kikuchi, Y. In Vivo Circular RNA Expression by the Permuted Intron-Exon Method. Innov. Biotechnol. 2012, 108 (4), 354– 356,  DOI: 10.5772/28220
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    There is no corresponding record for this reference.
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    Sprengart, M. L.; Fuchs, E.; Porter, A. G. The Downstream Box: An Efficient and Independent Translation Initiation Signal in Escherichia Coli. EMBO J. 1996, 15 (3), 665– 674,  DOI: 10.1002/j.1460-2075.1996.tb00399.x
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    The downstream box: an efficient and independent translation initiation signal in Escherichia coli
    Sprengart, Michael L.; Fuchs, Eckart; Porter, Alan G.
    EMBO Journal (1996), 15 (3), 665-74CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
    The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.
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    Etchegaray, J.-P.; Inouye, M. Translational Enhancement by an Element Downstream of the Initiation Codon in Escherichia Coli*. J. Biol. Chem. 1999, 274, 10079,  DOI: 10.1074/jbc.274.15.10079
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    Translational enhancement by an element downstream of the initiation codon in Escherichia coli
    Etchegaray, Jean-Pierre; Inouye, Masayori
    Journal of Biological Chemistry (1999), 274 (15), 10079-10085CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addn. to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.
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    Van Den Berg, S.; Löfdahl, P. Å.; Härd, T.; Berglund, H. Improved Solubility of TEV Protease by Directed Evolution. J. Biotechnol. 2006, 121 (3), 291– 298,  DOI: 10.1016/j.jbiotec.2005.08.006
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    Improved solubility of TEV protease by directed evolution
    van den Berg, Susanne; Loefdahl, Per-Aeke; Haerd, Torleif; Berglund, Helena
    Journal of Biotechnology (2006), 121 (3), 291-298CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the prodn. of TEV protease in E. coli is hampered by low soly. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of sol. protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purifn., or other applications. Fluorescence based in vivo soly. screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of sol. TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purifn., and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.
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    Kapust, R. B.; Tözsér, J.; Fox, J. D.; Anderson, D. E.; Cherry, S.; Copeland, T. D.; Waugh, D. S. Tobacco Etch Virus Protease: Mechanism of Autolysis and Rational Design of Stable Mutants with Wild-Type Catalytic Proficiency. Protein Eng., Des. Sel. 2001, 14 (12), 993– 1000,  DOI: 10.1093/protein/14.12.993
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    Cech, T. R. Self-Splicing of Group I Introns. Annu. Rev. Biochem. 1990, 59, 543– 568,  DOI: 10.1146/annurev.bi.59.070190.002551
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    Self-splicing of group I introns
    Cech, Thomas R.
    Annual Review of Biochemistry (1990), 59 (), 543-68CODEN: ARBOAW; ISSN:0066-4154.
    A review with 165 refs. on the reaction pathway of self-splicing of group I introns in RNA, structure of group I introns, enzymic reactions of the Tetrahymena intron, the chem. of the splicing reaction, protein facilitation of the splicing, and intron mobility.
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    Guo, F.; Gooding, A. R.; Cech, T. R. Structure of the Tetrahymena Ribozyme. Mol. Cell 2004, 16 (3), 351– 362,  DOI: 10.1016/j.molcel.2004.10.003
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    Structure of the Tetrahymena ribozyme: Base triple sandwich and metal ion at the active site
    Guo, Feng; Gooding, Anne R.; Cech, Thomas R.
    Molecular Cell (2004), 16 (3), 351-362CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
    The Tetrahymena intron is an RNA catalyst, or ribozyme. As part of its self-splicing reaction, this ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand. Here we report the refined crystal structure of an active Tetrahymena ribozyme in the absence of its RNA substrate at 3.8 Å resoln. The 3'-terminal guanosine (ωG), which serves as the attacking group for RNA cleavage, forms a coplanar base triple with the G264-C311 base pair, and this base triple is sandwiched by three other base triples. In addn., a metal ion is present in the active site, contacting or positioned close to the ribose of the ωG and five phosphates. All of these phosphates have been shown to be important for catalysis. Therefore, we provide a picture of how the ribozyme active site positions both a catalytic metal ion and the nucleophilic guanosine for catalysis prior to binding its RNA substrate.
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    Michel, F.; Hanna, M.; Green, R.; Bartel, D. P.; Szostak, J. W. The Guanosine Binding Site of the Tetrahymena Ribozyme. Nature 1989, 342 (6248), 391– 395,  DOI: 10.1038/342391a0
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    The guanosine binding site of the Tetrahymena ribozyme
    Michel, Francois; Hanna, Maya; Green, Rachel; Bartel, David P.; Szostak, Jack W.
    Nature (London, United Kingdom) (1989), 342 (6248), 391-5CODEN: NATUAS; ISSN:0028-0836.
    The self-splicing Group I introns have a highly specific binding site for the substrate guanosine. Mutant versions of the Tetrahymena ribozyme have been used in combination with guanosine analogs to identify the nucleotide in the ribozyme that is primarily responsible for recognition of the guanine base.
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    Legault, P.; Herschlag, D.; Celander, D. W.; Cech, T. R. Mutations at the Guanosine-Binding Site of the Tetrahymena Ribozyme Also Affect Site-Specific Hydrolysis. Nucleic Acids Res. 1992, 20 (24), 6613– 6619,  DOI: 10.1093/nar/20.24.6613
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    34
    Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis
    Legault, Pascale; Herschlag, Daniel; Celander, Daniel W.; Cech, Thomas R.
    Nucleic Acids Research (1992), 20 (24), 6613-19CODEN: NARHAD; ISSN:0305-1048.
    Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of anal. of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside). The effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 × 103 when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 × 105. The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concn. or lowering the temp. In addn., the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA it is concluded that the catalytic role of this conserved nucleotide is not limited to guanosine binding.
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  35. 35
    Varik, V.; Oliveira, S. R. A.; Hauryliuk, V.; Tenson, T. HPLC-Based Quantification of Bacterial Housekeeping Nucleotides and Alarmone Messengers PpGpp and PppGpp. Sci. Rep. 2017, 7 (1), 1– 12,  DOI: 10.1038/s41598-017-10988-6
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    35
    HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.
    Varik, Vallo; Oliveira, Sofia Raquel Alves; Hauryliuk, Vasili; Tenson, Tanel
    Scientific Reports (2017), 7 (1), 1-12CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved expts. feasible. The method combines cell harvesting by rapid filtration, followed by acid extn., freeze-drying with chromatog. sepn. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labeling with 32P-labeled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40- and =8-fold, resp.) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.
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  36. 36
    Chen, X.; Mohr, G.; Lambowitz, A. M. The Neurosporea Crassa CYT-18 Protein C-Terminal RNA-Binding Domain Helps Stabilize Interdomain Tertiary Interactions in Group I Introns. RNA 2004, 10 (4), 634– 644,  DOI: 10.1261/rna.5212604
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    36
    The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns
    Chen, Xin; Mohr, Georg; Lambowitz, Alan M.
    RNA (2004), 10 (4), 634-644CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate α-helical, and C-terminal RNA-binding domains that also function in binding tRNATyr. Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix contg. the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.
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  37. 37
    Wei, L.; Cai, X.; Qi, Z.; Rong, L.; Cheng, B.; Fan, J. In Vivo and in Vitro Characterization of TEV Protease Mutants. Protein Expression Purif. 2012, 83 (2), 157– 163,  DOI: 10.1016/j.pep.2012.03.011
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    37
    In vivo and in vitro characterization of TEV protease mutants
    Wei, Lingling; Cai, Xueyan; Qi, Zhenguo; Rong, Liang; Cheng, Beijiu; Fan, Jun
    Protein Expression and Purification (2012), 83 (2), 157-163CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
    Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, the prodn. of TEV protease in Escherichia coli is hampered by low yield and poor soly., and autocleavage of wild-type TEVp gives rise to loss-of-function. Previously it was reported that TEVp mutant S219V displayed more stability, and TEVp variant contg. T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced prodn. and soly., resp. Here, the authors introduced T17S/N68D/I77V mutations in TEVp S219V to generate TEVpM1 and combined 5 amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and 2 constructed variants, TEVpM2 displayed the highest soly. and catalytic activity in vivo, using EmGFP as the soly. reporter, and the designed fusion protein as in vivo substrate contg. an N-terminal hexahistidine-tagged glutathione S-transferase, a peptide sequence for thrombin and TEV cut and Eschericia coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tags at the N- and C-termini showed the highest yield, soly., and cleavage efficiency. Mutations of 5 amino acid residues in TEVpM2 slightly altered the protein secondary structure conformed by CD assay.
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  38. 38
    Fang, J.; Chen, L.; Cheng, B.; Fan, J. Engineering Soluble Tobacco Etch Virus Protease Accompanies the Loss of Stability. Protein Expression Purif. 2013, 92 (1), 29– 35,  DOI: 10.1016/j.pep.2013.08.015
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    Engineering soluble tobacco etch virus protease accompanies the loss of stability
    Fang, Jie; Chen, Ling; Cheng, Beijiu; Fan, Jun
    Protein Expression and Purification (2013), 92 (1), 29-35CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
    Tobacco etch virus protease (TEVp) is a widely used tool enzyme in biol. studies. To improve the soly. of recombinant TEVp, three variants, including the double mutant (L56V/S135G), the triple mutant (T17S/N68D/I77V), and the quintuple mutant (T17S/L56V/N68D/I77V/S135G), have been developed, however, with little information on functional stability. Here we investigated the soly. and stability of the three TEVp mutants under different temp. and denaturants, and in Escherichia coli with different cultural conditions. The quintuple mutant showed the highest soly. and thermostability, and the double mutant was most resistant to the denaturants. The double mutant folded best in E. coli cells at 37°C with or without the co-expressed mol. chaperones GroEL, GroES and GrpE. The least sol. wild type TEVp displayed better tolerance to denaturants than the triple and the quintuple mutants. All results demonstrated that TEVp is not engineered to embody the most desirable soly. and stability by the current mutations.
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  39. 39
    Sanchez, M. I.; Ting, A. Y. Directed Evolution Improves the Catalytic Efficiency of TEV Protease. Nat. Methods 2020, 17 (2), 167– 174,  DOI: 10.1038/s41592-019-0665-7
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    Directed evolution improves the catalytic efficiency of TEV protease
    Sanchez, Mateo I.; Ting, Alice Y.
    Nature Methods (2020), 17 (2), 167-174CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnol. because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1delta), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resoln. Given the widespread use of TEV in biotechnol., both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.
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  40. 40
    Colussi, T. M.; Costantino, D. A.; Zhu, J.; Donohue, J. P.; Korostelev, A. A.; Jaafar, Z. A.; Plank, T. D. M.; Noller, H. F.; Kieft, J. S. Initiation of Translation in Bacteria by a Structured Eukaryotic IRES RNA. Nature 2015, 519 (7541), 110– 113,  DOI: 10.1038/nature14219
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    Initiation of translation in bacteria by a structured eukaryotic IRES RNA
    Colussi, Timothy M.; Costantino, David A.; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A.; Jaafar, Zane A.; Plank, Terra-Dawn M.; Noller, Harry F.; Kieft, Jeffrey S.
    Nature (London, United Kingdom) (2015), 519 (7541), 110-113CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical mol. signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been obsd. in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resoln., revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by tRNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.
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    Litke, J. L.; Jaffrey, S. R. Highly Efficient Expression of Circular RNA Aptamers in Cells Using Autocatalytic Transcripts. Nat. Biotechnol. 2019, 37 (6), 667– 675,  DOI: 10.1038/s41587-019-0090-6
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    Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Litke, Jacob L.; Jaffrey, Samie R.
    Nature Biotechnology (2019), 37 (6), 667-675CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degrdn. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Addnl., an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
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    Levin-Karp, A.; Barenholz, U.; Bareia, T.; Dayagi, M.; Zelcbuch, L.; Antonovsky, N.; Noor, E.; Milo, R. Quantifying Translational Coupling in E. Coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters. ACS Synth. Biol. 2013, 2 (6), 327– 336,  DOI: 10.1021/sb400002n
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    Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters
    Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron
    ACS Synthetic Biology (2013), 2 (6), 327-336CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was obsd. in prokaryotic operons several decades ago, but the quant. range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Addnl., we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quant. characterize translational coupling in a synthetic E. coli operon. Our anal. will be useful in accurate manipulation of gene expression in synthetic biol. and serves as a step toward understanding the mechanisms involved in translational expression modulation.
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    Zhang, Y.; Chen, H.; Zhang, Y.; Yin, H.; Zhou, C.; Wang, Y. Direct RBS Engineering of the Biosynthetic Gene Cluster for Efficient Productivity of Violaceins in E. Coli. Microb. Cell Fact. 2021, 20 (1), 1– 13,  DOI: 10.1186/s12934-021-01518-1
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    Zhang, X.; Lin, Y.; Wu, Q.; Wang, Y.; Chen, G. Q. Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering. Trends Biotechnol. 2020, 38 (7), 689– 700,  DOI: 10.1016/j.tibtech.2019.10.006
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    Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering
    Zhang, Xu; Lin, Yina; Wu, Qiong; Wang, Ying; Chen, Guo-Qiang
    Trends in Biotechnology (2020), 38 (7), 689-700CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biol. and DNA-editing approaches have been adopted to engineer cells for more efficient PHA prodn. Recent advances in synthetic biol. applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphol. engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-assocd. protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biol. methods and genome-editing tools contribute to controllable PHA mol. wts. and compns., enhanced PHA accumulation, and easy downstream processing.
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    Sørensen, M. A.; Fricke, J.; Pedersen, S. Ribosomal Protein S1 Is Required for Translation of Most, It Not All, Natural MRNAs in Escherichia Coli in Vivo. J. Mol. Biol. 1998, 280 (4), 561– 569,  DOI: 10.1006/jmbi.1998.1909
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    Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo
    Sorensen, Michael A.; Fricke, Jens; Pedersen, Steen
    Journal of Molecular Biology (1998), 280 (4), 561-569CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    We have deleted the chromosomal rpsA gene, encoding ribosomal protein S1, from an Escherichia coli strain carrying a plasmid where rpsA was controlled by the lac promoter and operator. This exogenous source of protein S1 was essential for growth. Thus we have verified the abs. requirement for protein S1. To see if translation of individual mRNAs differed in the requirements for protein S1, we removed the inducer and followed the time-course of the synthesis of several individual proteins and of total RNA, DNA, and protein. Growth immediately shifted from being exponential to being linear, with a rate of protein synthesis defined by the pre-existing amt. of protein S1. The expression pattern of the individual proteins indicated that the translation of all mRNAs was dependent on protein S1. Unexpectedly, we found that depletion for protein S1 for extended periods introduced a starvation for amino acids. Such starvation was indicated by an increased synthesis of ppGpp and could be reversed by addn. of a mixt. of all 20 amino acids. Measurements of the peptide chain elongation rate in vivo showed that ribosomes without protein S1 were unable to interfere with the peptide chain elongation rate of the active ribosomes and that, therefore, protein S1 was unable to diffuse from one ribosome to another during translation. We conclude that protein S1-deficient ribosomes are totally inactive in peptide chain elongation on most, if not all, naturally occurring E. coli mRNAs. (c) 1998 Academic Press.
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    Boni, I. V.; Lsaeva, D. M.; Musychenko, M. L.; Tzareva, N. V. Ribosome-Messenger Recognition: MRNA Target Sites for Ribosomal Protein S1. Nucleic Acids Res. 1991, 19 (1), 155– 162,  DOI: 10.1093/nar/19.1.155
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    Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1
    Boni, I. V.; Isaeva, D. M.; Musychenko, M. L.; Tsareva, N. V.
    Nucleic Acids Research (1991), 19 (1), 155-62CODEN: NARHAD; ISSN:0305-1048.
    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. S1 binding sites were identified on several phage RNAs in preinitiation complexes using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradn. Targets for S1 on Qβ and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Qβ RNA, this S1 binding site overlaps the S-site for Qβ replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea, Escherichia coli ssb mRNA prep. was combined in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymic footprinting, and UV crosslinking, it was shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. It is proposed that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome.
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    Rasila, T. S.; Pajunen, M. I.; Savilahti, H. Critical Evaluation of Random Mutagenesis by Error-Prone Polymerase Chain Reaction Protocols, Escherichia Coli Mutator Strain, and Hydroxylamine Treatment. Anal. Biochem. 2009, 388 (1), 71– 80,  DOI: 10.1016/j.ab.2009.02.008
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    Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment
    Rasila, Tiina S.; Pajunen, Maria I.; Savilahti, Harri
    Analytical Biochemistry (2009), 388 (1), 71-80CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chem. and biol. methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions vs. transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended av. frequency of induced mutations can be adjusted to a desirable level.
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    McInerney, P.; Adams, P.; Hadi, M. Z. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Mol. Biol. Int. 2014, 2014, 1– 8,  DOI: 10.1155/2014/287430
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    Patrick, W. M.; Firth, A. E.; Blackburn, J. M. User-Friendly Algorithms for Estimating Completeness and Diversity in Randomized Protein-Encoding Libraries. Protein Eng., Des. Sel. 2003, 16 (6), 451– 457,  DOI: 10.1093/protein/gzg057
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    Vanhercke, T.; Ampe, C.; Tirry, L.; Denolf, P. Reducing Mutational Bias in Random Protein Libraries. Anal. Biochem. 2005, 339 (1), 9– 14,  DOI: 10.1016/j.ab.2004.11.032
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    Reducing mutational bias in random protein libraries
    Vanhercke, Thomas; Ampe, Christophe; Tirry, Luc; Denolf, Peter
    Analytical Biochemistry (2005), 339 (1), 9-14CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    The success of protein optimization through directed mol. evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by sep. error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate nos. of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concn. of template in the shuffling reaction while taking into account the GC content of the target gene.
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    Lee, S. O.; Fried, S. D. An Error-Prone PCR Method for Small Amplicons. Anal. Biochem. 2021, 628, 114266,  DOI: 10.1016/j.ab.2021.114266
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    An error prone PCR method for small amplicons
    Lee, Sea On; Fried, Stephen D.
    Analytical Biochemistry (2021), 628 (), 114266CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
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  1. Ghita Guessous, Lauren Blake, Anthony Bui, Yelim Woo, Gabriel Manzanarez. Disentangling the Web: An Interdisciplinary Review on the Potential and Feasibility of Spider Silk Bioproduction. ACS Biomaterials Science & Engineering 2024, 10 (9) , 5412-5438. https://doi.org/10.1021/acsbiomaterials.4c00145
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  4. Qi Xie, Sea On Lee, Nitya Vissamsetti, Sikao Guo, Margaret E. Johnson, Stephen D. Fried. Secretion‐Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface. Angewandte Chemie 2023, 135 (37) https://doi.org/10.1002/ange.202305178
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  • Abstract

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    Figure 1

    Figure 1. Approaches to generate tandem repeat proteins. (A) A plasmid encoding a monomeric unit is cut with two restriction enzymes to generate an insert. The original plasmid is cut with one of the two restriction enzymes to create self-complementary sticky ends, which allows ligation of the insert back into its original vector, creating a plasmid that encodes a tandem repeat. Upon ligation in a tail-to-head manner, the restriction site is destroyed, allowing directional cloning (inverse repeats can be removed by redigesting). This process can be repeated to generate larger 2n multimers. (B) A short segment of DNA encoding a monomeric unit is digested and circularized in vitro. The circular DNA serves as the template for rolling circle amplification and generates a mixture of concatemers with different lengths. The desired size of the repetitive sequences can be selected by gel extraction, digested, and cloned back into an expression vector. (C) In loopable translation, a plasmid is created in which a segment of DNA encoding a monomeric unit is incorporated within a permuted self-splicing Group I intron (gray blocks). The plasmid is transformed into an expression strain, and its gene is transcribed into RNA, which is circularized. The circular mRNA translates into a repetitive protein product through ribosome looping.

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    Figure 2

    Figure 2. Design of a loopable translator with a coupled fluorescence GFP reporter. (A) Sequence, secondary structure, and mechanism of the self-splicing Group I intron from the thymidylate synthase (td) gene of T4 bacteriophage. This intron catalyzes two consecutive, site-specific phosphoryl transfer reactions, which, in the natural form, results in the splicing of the two flanking exons (orange). The 5′ splice site (marked with a red arrow) is selected by base-pairing between the 5′ exonic sequence (5′ Ex) and an internal guide sequence (IGS) at the beginning of the intron (P1), while the 3′ splice site (marked with an orange arrow) is selected by a short 2 bp stem formed between the 3′ exonic sequence (3′ Ex) and the edge of the P1 loop (termed P10, shown by two gray lines). An exogenous guanosine (red) is required to initiate the reaction and becomes prepended to the 5′-terminus of the intron sequence (black). 5′* and 3′* denote the termini of the RNA molecule in its natural form. In our construct, the circular permutation in the ORF region of P6a results in two new termini (labeled 5′ and 3′), and a permuted GFP sequence (green) is incorporated between 3′* and 5′*. In this reorganized topology, intron activity will result in circularization of the internal “exonic” region. (B) Design of a GFP fluorescence reporter system for RNA circularization. A plasmid is created (pBAD-tdTEVDB) in which a permuted superfolder GFP (sfGFP) gene is incorporated within the permuted intron. The GFP is split such that the N-terminal portion (residues 1–52) is placed downstream of the C-terminal portion (residues 53–241). In between these two coding regions are inserted a TEV protease site and a ribosome binding site (RBS) along with an enhancing downstream box (DB) in frame with the GFP coding sequence. After intron folding and RNA circularization, an mRNA is formed which is competent to recruit ribosomes and generate full-length GFP. Because polymeric GFP is found to have low fluorescence, this system is coexpressed with TEV protease, which can liberate fluorescent GFP monomers from the primary chain product. (C) A negative control plasmid (pBAD-sfGFP1–52) encodes the protein product that would form in the absence of circularization (with the same promoter, origin, and selectable marker as pBAD-tdTEVDB). A positive control plasmid (pBAD-sfGFP) encodes the protein product that would form upon circularization. Importantly, it differs from a wild-type sfGFP in that it has a 10-residue long “scar sequence” in between residues 52 and 53, which result from exonic context sequences (represented as orange boxes) that were retained to ensure proper base-pairing with the IGSs. pBAD-tdTEVDB-STOP is identical to pBAD-tdTEVDB except that a stop codon is placed at the end of GFP. (D) pBAD-tdTEVDB generates a fluorescence signal that is significantly higher than the background level (P < 0.0001 by Student’s t test) but represents 4.2% of the signal of the corresponding positive control (n = 3). Fluorescence measurements were conducted in biological triplicate.

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    Figure 3

    Figure 3. High Mg and reduced temperature enhance loopable translation. (A) Bar chart showing the levels of fluorescence of several constructs (negative control (−), positive control (+), and pBAD-tdTEVDB) expressed at 37 °C with varying concentrations of guanosine supplemented to the growth media during expression assays. Additional guanosine had no significant effect on the fluorescence signal generated by pBAD-tdTEVDB. (B) Bar chart showing the levels of fluorescence of the same set of constructs expressed at 37 °C with varying concentrations of MgCl2 supplemented to the growth media during expressions assays. Higher MgCl2 concentrations had a beneficial effect; relative to 1 mM MgCl2, fluorescence levels in 20 mM MgCl2 were 1.2-fold higher (P-value = 0.01). (C) Bar chart showing the levels of fluorescence of the same set of constructs with varying MgCl2 supplemented to the growth media, but with expressions carried out at 30 °C instead of 37 °C. Lower temperature had a significantly beneficial effect on fluorescence signal (2.8-fold at 20 mM MgCl2, P-value <0.0001). Moreover, at the lower temperature, the fluorescence signal significantly benefitted from high concentrations of MgCl2 (a: 1.9-fold, P-value = 0.0006). Under these conditions, the loopable translator generated 16% of the fluorescence of the positive control. Fluorescence levels from pBAD-tdTEVDB-STOP (see Figure 2C, labeled “STOP”) were slightly higher than those from the negative control (b: 1.05-fold, P-value = 0.04), but much less than the loopable translator (c: 4.8-fold, P-value <0.0001). Fluorescence measurements were conducted in biological triplicate; statistical tests were conducted with Student’s t test.

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    Figure 4

    Figure 4. Verification of RNA circularization and polyGFP synthesis. (A) Anti-His Western blot image showing the protein products of pBAD-sfGFP, pBAD-tdTEVDB, pBAD-tdTEVDB-STOP, and pBAD-tdTEVDB-mCherry that were expressed with and without the pRK793 plasmid (expressing TEV protease). pBAD-tdTEVDB in the absence of TEV protease generated proteins of high molecular weight. Expression in the presence of TEV protease generates a species with the molecular weight of monomeric GFP (25 kDa). (B) Densitometry analysis showing average intensities relative to a positive control, for three biological replicates of the Western blot. (C) Construct maps of the positive control for the mCherry experiment (pBAD-sfGFP(52-DVFLGLPFNI)-mCherry), pBAD-tdTEVDB, and pBAD-tdTEVDB-mCherry. pBAD-tdTEVDB-mCherry was designed to form a larger mRNA loop compared to that of pBAD-tdTEVDB, in which GFP would be expressed only upon circularization, whereas mCherry would be expressed independently of circularization. (D) Bar chart showing the levels of fluorescence from the constructs shown in part C at 30 °C with varying concentrations of MgCl2. The positive control shows the native difference in fluorescence between GFP and mCherry and serves as a normalization factor. With pBAD-tdTEVDB-mCherry, 25% of the target mRNA achieved circularization at 20 mM Mg2+. The larger mRNA loop was detrimental for circularization, leading to lower levels of GFP fluorescence compared to those of pBAD-tdTEVDB (P = 0.0066 by Student’s t test).

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    Figure 5

    Figure 5. Minimal context requirements for loopable translator. (A) Secondary structure of the loopable translator highlighting the 10 amino acid “scar sequence” that is incorporated into the loop because of the inclusion of 30 nt of exonic context retained from the natural td gene (15 nt from the original 5′ exon, and 15 nt from the original 3′ exon). Each codon triplet is color coded, and deletions of this context sequence are represented through their corresponding color blocks in bar charts C–E. (B) Secondary structure that is formed when all 15 nt (coding for DVFLG) of the 5′ exonic context sequence are deleted. The nucleotides that correspond to residues 50–52 of GFP (green) replace the original 5′ exon to pair with the IGS. Nucleotides marked with * (in red) represent compensatory mutations in the P1 IGS (termed the modified P1*). (C) Bar chart showing the level of fluorescence from a truncation series in which the 5′ context sequence was deleted one codon at a time. (D) Bar chart showing the level of fluorescence from a truncation series in which the 3′ context sequence was deleted one codon at a time. (E) Finding the minimal context requirements. Overall, the entire 5′ context sequence can be deleted, and all but the last 3 nucleotides of the 3′ context sequence (which form P10) can be deleted. Strengthening P10 (P10*) did not have a beneficial effect. All fluorescence measurements were conducted in biological triplicate.

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    Figure 6

    Figure 6. Directed evolution on the initiation sequence of loopable translator with minimum context. (A) Error-prone PCR was performed on a short 36 bp amplicon encoding the initiation region [ribosome binding site (RBS), spacer, initiator methionine, and a downstream box (DB)] of the GFP reporter gene. (B) Bar chart showing the fluorescence signals from the top-performing constructs selected from the first round of directed evolution (n = 3 biological replicates following primary screen). A construct with a point mutation (G4C) generated a signal that is significantly higher than the wild-type (pBAD-tdTEVDB) (1.9-fold; P = 0.0065). (C) Histogram showing the fluorescence signal of 372 constructs screened in the first round of directed evolution relative to the wild-type. (D) Bar chart showing the fluorescence signal from the top-performing constructs selected from the second round of directed evolution (n = 3 biological replicates following primary screen). A construct with an additional mutation (C-15A/G4C) generated a signal that was ca. 3-fold higher than the wild-type (a: P = 0.0039) and ca. 2-fold higher than the single mutant (b: P = 0.033). (E) Histogram showing the fluorescence signal of 720 constructs screened in the second round of directed evolution relative to G4C. All statistical tests were conducted using Student’s t test

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    Figure 7

    Figure 7. Applying pBAD-tdTEVDB to producing spider silk spidroin. (A) Illustration of the nonlooped and looped constructs containing repetitive units of dragline silk. A repetitive unit with 36 amino acids was chosen as a monomeric unit. Six constructs were generated, comprising 8, 16, or 24 tandem repeats of a repetitive protein unit derived from major ampullate spidroin protein 1 (MaSp1), cloned into either a standard expression vector or the loopable translator. (B) Anti-His Western blot image demonstrating the protein products of the six constructs. Apparent low molecular weights from the loopable translator could be due to high insolubility and/or instability of the resulting protein products.

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  • References

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      A review. Curli are the major proteinaceous component of a complex extracellular matrix produced by many Enterobacteriaceae. Curli were first discovered in the late 1980s on Escherichia coli strains that caused bovine mastitis, and have since been implicated in many physiol. and pathogenic processes of E. coli and Salmonella spp. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation. Curli also mediate host cell adhesion and invasion, and they are potent inducers of the host inflammatory response. The structure and biogenesis of curli are unique among bacterial fibers that have been described to date. Structurally and biochem., curli belong to a growing class of fibers known as amyloids. Amyloid fiber formation is responsible for several human diseases including Alzheimer's, Huntington's, and prion diseases, although the process of in vivo amyloid formation is not well understood. Curli provide a unique system to study macromol. assembly in bacteria and in vivo amyloid fiber formation. Here, the authors review curli biogenesis, regulation, role in biofilm formation, and role in pathogenesis.
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      Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization
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      Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnol. to energy. However, it is sometimes challenging to assemble free short peptides into functional supramol. structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling β-helical subunit composed of five pseudorepeats (R1-R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphol. of the truncated and HAP-contg. curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocryst. HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVSmtbvJ&md5=27179a2b3ffc20d4a9d18b5f744ce6ef
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      Structural changes of TasA in biofilm formation of Bacillus subtilis
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      13
      Nanomechanics of functional and pathological amyloid materials
      Knowles, Tuomas P. J.; Buehler, Markus J.
      Nature Nanotechnology (2011), 6 (8), 469-479CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      A review. Amyloid or amyloid-like fibrils represent a general class of nanomaterials that can be formed from many different peptides and proteins. Although these structures have an important role in neurodegenerative disorders, amyloid materials have also been exploited for functional purposes by organisms ranging from bacteria to mammals. Here we review the functional and pathol. roles of amyloid materials and discuss how they can be linked back to their nanoscale origins in the structure and nanomechanics of these materials. We focus on insights both from expts. and simulations, and discuss how comparisons between functional protein filaments and structures that are assembled abnormally can shed light on the fundamental material selection criteria that lead to evolutionary bias in multiscale material design in nature.
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    14. 14
      Vural, M.; Lei, Y.; Pena-Francesch, A.; Jung, H.; Allen, B.; Terrones, M.; Demirel, M. C. Programmable Molecular Composites of Tandem Proteins with Graphene Oxide for Efficient Bimorph Actuators. Carbon 2017, 118, 404– 412,  DOI: 10.1016/j.carbon.2017.03.053
      14
      Programmable molecular composites of tandem proteins with graphene oxide for efficient bimorph actuators
      Vural, Mert; Lei, Yu; Pena-Francesch, Abdon; Jung, Huihun; Allen, Benjamin; Terrones, Mauricio; Demirel, Melik C.
      Carbon (2017), 118 (), 404-412CODEN: CRBNAH; ISSN:0008-6223. (Elsevier Ltd.)
      The rapid expansion in the spectrum of two-dimensional (2D) materials has driven research efforts on the fabrication of 2D composites and heterostructures. Highly ordered structure of 2D materials provides an excellent platform for controlling the ultimate structure and properties of the composite material with precision. However, limited control over the structure of the matrix phase and its interactions with highly ordered 2D materials results in defective composites with inferior performance. Here, we demonstrate the successful synthesis, integration, and characterization of hybrid 2D material systems consisting of tandem repeat (TR) proteins inspired by squid ring teeth and graphene oxide (GO). The TR protein layer acts as a unique programmable assembler for GO layers with precise control of interlayer distance of less than 1 nm. As an application, we further demonstrate thermal actuation using bimorph mol. composite films. Bimorph actuators made of mol. composite films (GO/TR) can lead to energy efficiencies 18 times higher than regular bimorph actuators consisting of a GO layer and a TR protein layer (i.e., conventional bulk composite of GO and TR). Addnl., mol. composite bimorph actuators can reach curvature values as high as 1.2 cm-1 by using TR proteins with higher mol. wt., which is 3 times higher than conventional GO and TR composites.
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    15. 15
      Pena-Francesch, A.; Akgun, B.; Miserez, A.; Zhu, W.; Gao, H.; Demirel, M. C. Pressure Sensitive Adhesion of an Elastomeric Protein Complex Extracted from Squid Ring Teeth. Adv. Funct. Mater. 2014, 24 (39), 6227– 6233,  DOI: 10.1002/adfm.201401534
      15
      Pressure Sensitive Adhesion of an Elastomeric Protein Complex Extracted From Squid Ring Teeth
      Pena-Francesch, Abdon; Akgun, Bulent; Miserez, Ali; Zhu, Wenpeng; Gao, Huajian; Demirel, Melik C.
      Advanced Functional Materials (2014), 24 (39), 6227-6233CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The pressure sensitive adhesion characteristic of a protein complex extd. from squid ring teeth (SRT), which exhibits an unusual and reversible transition from a solid to a melt, is studied. The native SRT is an elastomeric protein complex that has std. amino acids, and it does not function as adhesives in nature. The SRT can be thermally shaped into any 3D geometry (e.g., thin films, ribbons, colloids), and it has a glass transition temp. of 32 °C in water. Underwater adhesion strength of the protein film is approx. 1.5-2.5 MPa. The thermoplastic protein film could potentially be used in an array of fields, including dental resins, bandages for wound healing, and surgical sutures in the body.
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    16. 16
      Pena-Francesch, A.; Jung, H.; Segad, M.; Colby, R. H.; Allen, B. D.; Demirel, M. C. Mechanical Properties of Tandem-Repeat Proteins Are Governed by Network Defects. ACS Biomater. Sci. Eng. 2018, 4 (3), 884– 891,  DOI: 10.1021/acsbiomaterials.7b00830
      16
      Mechanical Properties of Tandem-Repeat Proteins Are Governed by Network Defects
      Pena-Francesch, Abdon; Jung, Huihun; Segad, Mo; Colby, Ralph H.; Allen, Benjamin D.; Demirel, Melik C.
      ACS Biomaterials Science & Engineering (2018), 4 (3), 884-891CODEN: ABSEBA; ISSN:2373-9878. (American Chemical Society)
      Topol. defects in highly repetitive structural proteins strongly affect their mech. properties. However, there are no universal rules for structure-property prediction in structural proteins due to high diversity in their repetitive modules. Here, we studied the mech. properties of tandem-repeat proteins inspired by squid-ring-teeth proteins using rheol. and tensile expts. as well as spectroscopic and x-ray techniques. We also developed a network model based on entropic elasticity to predict structure-property relations for these proteins. We demonstrated that shear modulus, elastic modulus, and toughness scaled inversely with the no. of repeats in these proteins. Through optimization of structural repeats, we obtained highly efficient protein network topologies with 40 MPa ultimate strength that were capable of withstanding deformations up to 400%. The investigation of topol. network defects in structural proteins will improve the prediction of mech. properties for designing novel protein-based materials.
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    17. 17
      Bale, J. B.; Gonen, S.; Liu, Y.; Shedffler, W.; King, N. P.; Baker, D. Accurate Design of Megadalton-Scale Two-Component Icosahedral Protein Complexes. Science (Washington, DC, U. S.) 2016, 353 (6297), 389– 394,  DOI: 10.1126/science.aaf8818
      17
      Accurate design of megadalton-scale two-component icosahedral protein complexes
      Bale, Jacob B.; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O.; Gonen, Tamir; King, Neil P.; Baker, David
      Science (Washington, DC, United States) (2016), 353 (6297), 389-394CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Nature provides many examples of self- and co-assembling protein-based mol. machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochem. reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, the authors report the computational design and exptl. characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with mol. wts. (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nm in diam.) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallog. show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of mol. cargo through charge complementarity. The ability to design megadalton-scale materials with at.-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based mol. machines.
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    18. 18
      King, N. P.; Jacobitz, A. W.; Sawaya, M. R.; Goldschmidt, L.; Yeates, T. O. Structure and Folding of a Designed Knotted Protein. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (48), 20732– 20737,  DOI: 10.1073/pnas.1007602107
      18
      Structure and folding of a designed knotted protein
      King, Neil P.; Jacobitz, Alex W.; Sawaya, Michael R.; Goldschmidt, Lukasz; Yeates, Todd O.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (48), 20732-20737, S20732/1-S20732/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A very small no. of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophys. characterization and x-ray crystal structure detn. show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approx. 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic expts. suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy - connecting two monomers of an intertwined homodimer into a single protein chain - supports a model for evolution of knotted structures via gene duplication.
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    19. 19
      Hershewe, J. M.; Wiseman, W. D.; Kath, J. E.; Buck, C. C.; Gupta, M. K.; Dennis, P. B.; Naik, R. R.; Jewett, M. C. Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12. ACS Synth. Biol. 2020, 9 (12), 3388– 3399,  DOI: 10.1021/acssynbio.0c00442
      19
      Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12
      Hershewe, Jasmine M.; Wiseman, William D.; Kath, James E.; Buck, Chelsea C.; Gupta, Maneesh K.; Dennis, Patrick B.; Naik, Rajesh R.; Jewett, Michael C.
      ACS Synthetic Biology (2020), 9 (12), 3388-3399CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramol. networks of nanoconfined β-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concn. on S12 soly., secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ~ 100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and β-sheet content by PEGylation. This work presents new insights into the biochem. of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.
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    20. 20
      Votteler, J.; Ogohara, C.; Yi, S.; Hsia, Y.; Nattermann, U.; Belnap, D. M.; King, N. P.; Sundquist, W. I. Designed Proteins Induce the Formation of Nanocage-Containing Extracellular Vesicles. Nature 2016, 540 (7632), 292– 295,  DOI: 10.1038/nature20607
      20
      Designed proteins induce the formation of nanocage-containing extracellular vesicles
      Votteler, Jorg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.
      Nature (London, United Kingdom) (2016), 540 (7632), 292-295CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Complex biol. processes are often performed by self-organizing nanostructures comprising multiple classes of macromols., such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biol. materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochem. analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (∼100 nm) vesicles contg. multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biol. materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer mols. between cells.
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    21. 21
      Jung, H.; Pena-Francesch, A.; Saadat, A.; Sebastian, A.; Kim, D. H.; Hamilton, R. F.; Albert, I.; Allen, B. D.; Demirel, M. C. Molecular Tandem Repeat Strategy for Elucidating Mechanical Properties of High-Strength Proteins. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (23), 6478– 6483,  DOI: 10.1073/pnas.1521645113
      21
      Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins
      Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (23), 6478-6483CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mech. properties remains elusive. We propose a new approach for the design and prodn. of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and cryst. domains. We produced segmented polypeptides with varying repeat no., while keeping the lengths and compns. of the amorphous and cryst. regions fixed. We showed that mech. properties of these synthetic proteins could be tuned by modulating their mol. wts. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the no. of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.
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    22. 22
      Ford, E.; Ares, M., Jr Synthesis of Circular RNA in Bacteria and Yeast Using RNA Cyclase Ribozymes Derived from a Group I Intron of Phage T4. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (8), 3117– 3121,  DOI: 10.1073/pnas.91.8.3117
      22
      Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4
      Ford, Ethan; Ares, Manuel, Jr.
      Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (8), 3117-21CODEN: PNASA6; ISSN:0027-8424.
      Studies on the function of circular RAN and RNA topol. in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). The authors show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, contg. 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topol., efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.
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    23. 23
      Perriman, R.; Ares, M. Circular MRNA Can Direct Translation of Extremely Long Repeating- Sequence Proteins in Vivo. RNA 1998, 4 (9), 1047– 1054,  DOI: 10.1017/S135583829898061X
      23
      Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo
      Perriman, Rhonda; Ares, Manuel, Jr.
      RNA (1998), 4 (9), 1047-1054CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant prodn. of such proteins for structural and engineering studies, we have produced circular mRNAs with infinite open reading frames. We show that a circular mRNA contg. a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Anal. of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technol. provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.
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    24. 24
      Perriman, R. Circular MRNA Encoding for Monomeric and Polymeric Green Fluorescent Protein. Methods Mol. Biol. 2002, 183, 69– 85,  DOI: 10.1385/1-59259-280-5:069
      24
      Circular mRNA encoding for monomeric and polymeric green fluorescent protein
      Perriman, Rhonda
      Methods in Molecular Biology (Totowa, NJ, United States) (2002), 183 (Green Fluorescent Protein), 69-85CODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)
      To facilitate recombinant prodn. of very long repeating proteins (e.g., silks, mollusk shell framework, etc.), the author has developed a method for producing mRNAs on circular RNA templates. This circularization process is derived from a rearranged group I intron, from which circular RNA is produced through the splicing activity of autocatalytic group I RNA elements. Because the only cofactors required for spicing of the group I intron are magnesium and guanosine, the process can take place in a variety of organisms, making it amenable to a wide variety of protein expression systems. This chapter details the design and construction of circular RNAs contg. the open reading frame encoding for green fluorescent protein (GFP). Included on the circular GFP mRNA constructs are translation initiation sequences designed to recruit either prokaryotic or eukaryotic ribosomes. The mRNAs produce extremely long protein chains of polyGFP, demonstrating that both prokaryotic and eukaryotic ribosomes can internally initiate and repeatedly transit a circular mRNA. Protocols are presented for constructing plasmids for prodn. of circular mRNA, for testing them, and for expressing the protein in Escherichia coli and in rabbit reticulocyte lysates.
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    25. 25
      Shub, D. A.; Gott, J. M.; Xu, M. Q.; Lang, B. F.; Michel, F.; Tomaschewski, J.; Pedersen-Lane, J.; Belfort, M. Structural Conservation among Three Homologous Introns of Bacteriophage T4 and the Group I Introns of Eukaryotes. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (4), 1151– 1155,  DOI: 10.1073/pnas.85.4.1151
      25
      Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes
      Shub, David A.; Gott, Jonatha M.; Xu, Ming Qun; Lang, B. Franz; Michel, Francois; Tomaschewski, Joerg; Pedersen-Lane, Joan; Belfort, Marlene
      Proceedings of the National Academy of Sciences of the United States of America (1988), 85 (4), 1151-5CODEN: PNASA6; ISSN:0027-8424.
      Three group I introns of phage T4 were compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the 2 newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, contg. phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the 3 introns. All 3 introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all 3 ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.
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    26. 26
      Umekage, S.; Uehara, T.; Fujita, Y.; Suzuki, H.; Kikuchi, Y. In Vivo Circular RNA Expression by the Permuted Intron-Exon Method. Innov. Biotechnol. 2012, 108 (4), 354– 356,  DOI: 10.5772/28220
      There is no corresponding record for this reference.
    27. 27
      Sprengart, M. L.; Fuchs, E.; Porter, A. G. The Downstream Box: An Efficient and Independent Translation Initiation Signal in Escherichia Coli. EMBO J. 1996, 15 (3), 665– 674,  DOI: 10.1002/j.1460-2075.1996.tb00399.x
      27
      The downstream box: an efficient and independent translation initiation signal in Escherichia coli
      Sprengart, Michael L.; Fuchs, Eckart; Porter, Alan G.
      EMBO Journal (1996), 15 (3), 665-74CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
      The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.
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    28. 28
      Etchegaray, J.-P.; Inouye, M. Translational Enhancement by an Element Downstream of the Initiation Codon in Escherichia Coli*. J. Biol. Chem. 1999, 274, 10079,  DOI: 10.1074/jbc.274.15.10079
      28
      Translational enhancement by an element downstream of the initiation codon in Escherichia coli
      Etchegaray, Jean-Pierre; Inouye, Masayori
      Journal of Biological Chemistry (1999), 274 (15), 10079-10085CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addn. to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.
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    29. 29
      Van Den Berg, S.; Löfdahl, P. Å.; Härd, T.; Berglund, H. Improved Solubility of TEV Protease by Directed Evolution. J. Biotechnol. 2006, 121 (3), 291– 298,  DOI: 10.1016/j.jbiotec.2005.08.006
      29
      Improved solubility of TEV protease by directed evolution
      van den Berg, Susanne; Loefdahl, Per-Aeke; Haerd, Torleif; Berglund, Helena
      Journal of Biotechnology (2006), 121 (3), 291-298CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the prodn. of TEV protease in E. coli is hampered by low soly. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of sol. protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purifn., or other applications. Fluorescence based in vivo soly. screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of sol. TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purifn., and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlvVGjsg%253D%253D&md5=3dadbdfff9ce324d2567b8f9a5f6abd0
    30. 30
      Kapust, R. B.; Tözsér, J.; Fox, J. D.; Anderson, D. E.; Cherry, S.; Copeland, T. D.; Waugh, D. S. Tobacco Etch Virus Protease: Mechanism of Autolysis and Rational Design of Stable Mutants with Wild-Type Catalytic Proficiency. Protein Eng., Des. Sel. 2001, 14 (12), 993– 1000,  DOI: 10.1093/protein/14.12.993
      There is no corresponding record for this reference.
    31. 31
      Cech, T. R. Self-Splicing of Group I Introns. Annu. Rev. Biochem. 1990, 59, 543– 568,  DOI: 10.1146/annurev.bi.59.070190.002551
      31
      Self-splicing of group I introns
      Cech, Thomas R.
      Annual Review of Biochemistry (1990), 59 (), 543-68CODEN: ARBOAW; ISSN:0066-4154.
      A review with 165 refs. on the reaction pathway of self-splicing of group I introns in RNA, structure of group I introns, enzymic reactions of the Tetrahymena intron, the chem. of the splicing reaction, protein facilitation of the splicing, and intron mobility.
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    32. 32
      Guo, F.; Gooding, A. R.; Cech, T. R. Structure of the Tetrahymena Ribozyme. Mol. Cell 2004, 16 (3), 351– 362,  DOI: 10.1016/j.molcel.2004.10.003
      32
      Structure of the Tetrahymena ribozyme: Base triple sandwich and metal ion at the active site
      Guo, Feng; Gooding, Anne R.; Cech, Thomas R.
      Molecular Cell (2004), 16 (3), 351-362CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
      The Tetrahymena intron is an RNA catalyst, or ribozyme. As part of its self-splicing reaction, this ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand. Here we report the refined crystal structure of an active Tetrahymena ribozyme in the absence of its RNA substrate at 3.8 Å resoln. The 3'-terminal guanosine (ωG), which serves as the attacking group for RNA cleavage, forms a coplanar base triple with the G264-C311 base pair, and this base triple is sandwiched by three other base triples. In addn., a metal ion is present in the active site, contacting or positioned close to the ribose of the ωG and five phosphates. All of these phosphates have been shown to be important for catalysis. Therefore, we provide a picture of how the ribozyme active site positions both a catalytic metal ion and the nucleophilic guanosine for catalysis prior to binding its RNA substrate.
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    33. 33
      Michel, F.; Hanna, M.; Green, R.; Bartel, D. P.; Szostak, J. W. The Guanosine Binding Site of the Tetrahymena Ribozyme. Nature 1989, 342 (6248), 391– 395,  DOI: 10.1038/342391a0
      33
      The guanosine binding site of the Tetrahymena ribozyme
      Michel, Francois; Hanna, Maya; Green, Rachel; Bartel, David P.; Szostak, Jack W.
      Nature (London, United Kingdom) (1989), 342 (6248), 391-5CODEN: NATUAS; ISSN:0028-0836.
      The self-splicing Group I introns have a highly specific binding site for the substrate guanosine. Mutant versions of the Tetrahymena ribozyme have been used in combination with guanosine analogs to identify the nucleotide in the ribozyme that is primarily responsible for recognition of the guanine base.
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    34. 34
      Legault, P.; Herschlag, D.; Celander, D. W.; Cech, T. R. Mutations at the Guanosine-Binding Site of the Tetrahymena Ribozyme Also Affect Site-Specific Hydrolysis. Nucleic Acids Res. 1992, 20 (24), 6613– 6619,  DOI: 10.1093/nar/20.24.6613
      34
      Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis
      Legault, Pascale; Herschlag, Daniel; Celander, Daniel W.; Cech, Thomas R.
      Nucleic Acids Research (1992), 20 (24), 6613-19CODEN: NARHAD; ISSN:0305-1048.
      Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of anal. of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside). The effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 × 103 when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 × 105. The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concn. or lowering the temp. In addn., the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA it is concluded that the catalytic role of this conserved nucleotide is not limited to guanosine binding.
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    35. 35
      Varik, V.; Oliveira, S. R. A.; Hauryliuk, V.; Tenson, T. HPLC-Based Quantification of Bacterial Housekeeping Nucleotides and Alarmone Messengers PpGpp and PppGpp. Sci. Rep. 2017, 7 (1), 1– 12,  DOI: 10.1038/s41598-017-10988-6
      35
      HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.
      Varik, Vallo; Oliveira, Sofia Raquel Alves; Hauryliuk, Vasili; Tenson, Tanel
      Scientific Reports (2017), 7 (1), 1-12CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved expts. feasible. The method combines cell harvesting by rapid filtration, followed by acid extn., freeze-drying with chromatog. sepn. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labeling with 32P-labeled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40- and =8-fold, resp.) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.
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    36. 36
      Chen, X.; Mohr, G.; Lambowitz, A. M. The Neurosporea Crassa CYT-18 Protein C-Terminal RNA-Binding Domain Helps Stabilize Interdomain Tertiary Interactions in Group I Introns. RNA 2004, 10 (4), 634– 644,  DOI: 10.1261/rna.5212604
      36
      The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns
      Chen, Xin; Mohr, Georg; Lambowitz, Alan M.
      RNA (2004), 10 (4), 634-644CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate α-helical, and C-terminal RNA-binding domains that also function in binding tRNATyr. Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix contg. the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.
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    37. 37
      Wei, L.; Cai, X.; Qi, Z.; Rong, L.; Cheng, B.; Fan, J. In Vivo and in Vitro Characterization of TEV Protease Mutants. Protein Expression Purif. 2012, 83 (2), 157– 163,  DOI: 10.1016/j.pep.2012.03.011
      37
      In vivo and in vitro characterization of TEV protease mutants
      Wei, Lingling; Cai, Xueyan; Qi, Zhenguo; Rong, Liang; Cheng, Beijiu; Fan, Jun
      Protein Expression and Purification (2012), 83 (2), 157-163CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
      Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, the prodn. of TEV protease in Escherichia coli is hampered by low yield and poor soly., and autocleavage of wild-type TEVp gives rise to loss-of-function. Previously it was reported that TEVp mutant S219V displayed more stability, and TEVp variant contg. T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced prodn. and soly., resp. Here, the authors introduced T17S/N68D/I77V mutations in TEVp S219V to generate TEVpM1 and combined 5 amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and 2 constructed variants, TEVpM2 displayed the highest soly. and catalytic activity in vivo, using EmGFP as the soly. reporter, and the designed fusion protein as in vivo substrate contg. an N-terminal hexahistidine-tagged glutathione S-transferase, a peptide sequence for thrombin and TEV cut and Eschericia coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tags at the N- and C-termini showed the highest yield, soly., and cleavage efficiency. Mutations of 5 amino acid residues in TEVpM2 slightly altered the protein secondary structure conformed by CD assay.
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    38. 38
      Fang, J.; Chen, L.; Cheng, B.; Fan, J. Engineering Soluble Tobacco Etch Virus Protease Accompanies the Loss of Stability. Protein Expression Purif. 2013, 92 (1), 29– 35,  DOI: 10.1016/j.pep.2013.08.015
      38
      Engineering soluble tobacco etch virus protease accompanies the loss of stability
      Fang, Jie; Chen, Ling; Cheng, Beijiu; Fan, Jun
      Protein Expression and Purification (2013), 92 (1), 29-35CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
      Tobacco etch virus protease (TEVp) is a widely used tool enzyme in biol. studies. To improve the soly. of recombinant TEVp, three variants, including the double mutant (L56V/S135G), the triple mutant (T17S/N68D/I77V), and the quintuple mutant (T17S/L56V/N68D/I77V/S135G), have been developed, however, with little information on functional stability. Here we investigated the soly. and stability of the three TEVp mutants under different temp. and denaturants, and in Escherichia coli with different cultural conditions. The quintuple mutant showed the highest soly. and thermostability, and the double mutant was most resistant to the denaturants. The double mutant folded best in E. coli cells at 37°C with or without the co-expressed mol. chaperones GroEL, GroES and GrpE. The least sol. wild type TEVp displayed better tolerance to denaturants than the triple and the quintuple mutants. All results demonstrated that TEVp is not engineered to embody the most desirable soly. and stability by the current mutations.
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    39. 39
      Sanchez, M. I.; Ting, A. Y. Directed Evolution Improves the Catalytic Efficiency of TEV Protease. Nat. Methods 2020, 17 (2), 167– 174,  DOI: 10.1038/s41592-019-0665-7
      39
      Directed evolution improves the catalytic efficiency of TEV protease
      Sanchez, Mateo I.; Ting, Alice Y.
      Nature Methods (2020), 17 (2), 167-174CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnol. because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1delta), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resoln. Given the widespread use of TEV in biotechnol., both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.
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    40. 40
      Colussi, T. M.; Costantino, D. A.; Zhu, J.; Donohue, J. P.; Korostelev, A. A.; Jaafar, Z. A.; Plank, T. D. M.; Noller, H. F.; Kieft, J. S. Initiation of Translation in Bacteria by a Structured Eukaryotic IRES RNA. Nature 2015, 519 (7541), 110– 113,  DOI: 10.1038/nature14219
      40
      Initiation of translation in bacteria by a structured eukaryotic IRES RNA
      Colussi, Timothy M.; Costantino, David A.; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A.; Jaafar, Zane A.; Plank, Terra-Dawn M.; Noller, Harry F.; Kieft, Jeffrey S.
      Nature (London, United Kingdom) (2015), 519 (7541), 110-113CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical mol. signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been obsd. in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resoln., revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by tRNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.
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    41. 41
      Litke, J. L.; Jaffrey, S. R. Highly Efficient Expression of Circular RNA Aptamers in Cells Using Autocatalytic Transcripts. Nat. Biotechnol. 2019, 37 (6), 667– 675,  DOI: 10.1038/s41587-019-0090-6
      41
      Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
      Litke, Jacob L.; Jaffrey, Samie R.
      Nature Biotechnology (2019), 37 (6), 667-675CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degrdn. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Addnl., an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
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    42. 42
      Levin-Karp, A.; Barenholz, U.; Bareia, T.; Dayagi, M.; Zelcbuch, L.; Antonovsky, N.; Noor, E.; Milo, R. Quantifying Translational Coupling in E. Coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters. ACS Synth. Biol. 2013, 2 (6), 327– 336,  DOI: 10.1021/sb400002n
      42
      Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters
      Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron
      ACS Synthetic Biology (2013), 2 (6), 327-336CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was obsd. in prokaryotic operons several decades ago, but the quant. range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Addnl., we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quant. characterize translational coupling in a synthetic E. coli operon. Our anal. will be useful in accurate manipulation of gene expression in synthetic biol. and serves as a step toward understanding the mechanisms involved in translational expression modulation.
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    43. 43
      Zhang, Y.; Chen, H.; Zhang, Y.; Yin, H.; Zhou, C.; Wang, Y. Direct RBS Engineering of the Biosynthetic Gene Cluster for Efficient Productivity of Violaceins in E. Coli. Microb. Cell Fact. 2021, 20 (1), 1– 13,  DOI: 10.1186/s12934-021-01518-1
      There is no corresponding record for this reference.
    44. 44
      Zhang, X.; Lin, Y.; Wu, Q.; Wang, Y.; Chen, G. Q. Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering. Trends Biotechnol. 2020, 38 (7), 689– 700,  DOI: 10.1016/j.tibtech.2019.10.006
      44
      Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering
      Zhang, Xu; Lin, Yina; Wu, Qiong; Wang, Ying; Chen, Guo-Qiang
      Trends in Biotechnology (2020), 38 (7), 689-700CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biol. and DNA-editing approaches have been adopted to engineer cells for more efficient PHA prodn. Recent advances in synthetic biol. applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphol. engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-assocd. protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biol. methods and genome-editing tools contribute to controllable PHA mol. wts. and compns., enhanced PHA accumulation, and easy downstream processing.
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    45. 45
      Sørensen, M. A.; Fricke, J.; Pedersen, S. Ribosomal Protein S1 Is Required for Translation of Most, It Not All, Natural MRNAs in Escherichia Coli in Vivo. J. Mol. Biol. 1998, 280 (4), 561– 569,  DOI: 10.1006/jmbi.1998.1909
      45
      Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo
      Sorensen, Michael A.; Fricke, Jens; Pedersen, Steen
      Journal of Molecular Biology (1998), 280 (4), 561-569CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      We have deleted the chromosomal rpsA gene, encoding ribosomal protein S1, from an Escherichia coli strain carrying a plasmid where rpsA was controlled by the lac promoter and operator. This exogenous source of protein S1 was essential for growth. Thus we have verified the abs. requirement for protein S1. To see if translation of individual mRNAs differed in the requirements for protein S1, we removed the inducer and followed the time-course of the synthesis of several individual proteins and of total RNA, DNA, and protein. Growth immediately shifted from being exponential to being linear, with a rate of protein synthesis defined by the pre-existing amt. of protein S1. The expression pattern of the individual proteins indicated that the translation of all mRNAs was dependent on protein S1. Unexpectedly, we found that depletion for protein S1 for extended periods introduced a starvation for amino acids. Such starvation was indicated by an increased synthesis of ppGpp and could be reversed by addn. of a mixt. of all 20 amino acids. Measurements of the peptide chain elongation rate in vivo showed that ribosomes without protein S1 were unable to interfere with the peptide chain elongation rate of the active ribosomes and that, therefore, protein S1 was unable to diffuse from one ribosome to another during translation. We conclude that protein S1-deficient ribosomes are totally inactive in peptide chain elongation on most, if not all, naturally occurring E. coli mRNAs. (c) 1998 Academic Press.
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    46. 46
      Boni, I. V.; Lsaeva, D. M.; Musychenko, M. L.; Tzareva, N. V. Ribosome-Messenger Recognition: MRNA Target Sites for Ribosomal Protein S1. Nucleic Acids Res. 1991, 19 (1), 155– 162,  DOI: 10.1093/nar/19.1.155
      46
      Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1
      Boni, I. V.; Isaeva, D. M.; Musychenko, M. L.; Tsareva, N. V.
      Nucleic Acids Research (1991), 19 (1), 155-62CODEN: NARHAD; ISSN:0305-1048.
      Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. S1 binding sites were identified on several phage RNAs in preinitiation complexes using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradn. Targets for S1 on Qβ and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Qβ RNA, this S1 binding site overlaps the S-site for Qβ replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea, Escherichia coli ssb mRNA prep. was combined in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymic footprinting, and UV crosslinking, it was shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. It is proposed that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome.
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    47. 47
      Rasila, T. S.; Pajunen, M. I.; Savilahti, H. Critical Evaluation of Random Mutagenesis by Error-Prone Polymerase Chain Reaction Protocols, Escherichia Coli Mutator Strain, and Hydroxylamine Treatment. Anal. Biochem. 2009, 388 (1), 71– 80,  DOI: 10.1016/j.ab.2009.02.008
      47
      Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment
      Rasila, Tiina S.; Pajunen, Maria I.; Savilahti, Harri
      Analytical Biochemistry (2009), 388 (1), 71-80CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chem. and biol. methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions vs. transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended av. frequency of induced mutations can be adjusted to a desirable level.
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    48. 48
      McInerney, P.; Adams, P.; Hadi, M. Z. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Mol. Biol. Int. 2014, 2014, 1– 8,  DOI: 10.1155/2014/287430
      There is no corresponding record for this reference.
    49. 49
      Patrick, W. M.; Firth, A. E.; Blackburn, J. M. User-Friendly Algorithms for Estimating Completeness and Diversity in Randomized Protein-Encoding Libraries. Protein Eng., Des. Sel. 2003, 16 (6), 451– 457,  DOI: 10.1093/protein/gzg057
      There is no corresponding record for this reference.
    50. 50
      Vanhercke, T.; Ampe, C.; Tirry, L.; Denolf, P. Reducing Mutational Bias in Random Protein Libraries. Anal. Biochem. 2005, 339 (1), 9– 14,  DOI: 10.1016/j.ab.2004.11.032
      50
      Reducing mutational bias in random protein libraries
      Vanhercke, Thomas; Ampe, Christophe; Tirry, Luc; Denolf, Peter
      Analytical Biochemistry (2005), 339 (1), 9-14CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      The success of protein optimization through directed mol. evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by sep. error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate nos. of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concn. of template in the shuffling reaction while taking into account the GC content of the target gene.
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    51. 51
      Lee, S. O.; Fried, S. D. An Error-Prone PCR Method for Small Amplicons. Anal. Biochem. 2021, 628, 114266,  DOI: 10.1016/j.ab.2021.114266
      51
      An error prone PCR method for small amplicons
      Lee, Sea On; Fried, Stephen D.
      Analytical Biochemistry (2021), 628 (), 114266CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      Error-prone PCR (epPCR) is a commonly employed approach in mol. biol., esp. in directed evolution, to generate libraries of DNA mols. with broad mutational spectrums. Though commonly applied to mutagenize protein coding sequences of several hundreds or thousands of basepairs, we found that commonly used protocols were not suitable for small (<100 bp) amplicons. Here we report a modified error-prone PCR protocol utilizing a Touchdown approach and employing only com. available components, that should be broadly useful for the researcher interested in concg. mutations into a small region of plasmid DNA. It will also be useful for achieving very high mutational loads on a std.-sized amplicon.
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    52. 52
      Farabaugh, P. J.; Björk, G. R. How Translational Accuracy Influences Reading Frame Maintenance. EMBO J. 1999, 18 (6), 1427– 1434,  DOI: 10.1093/emboj/18.6.1427
      52
      How translational accuracy influences reading frame maintenance
      Farabaugh, Philip J.; Bjork, Glenn R.
      EMBO Journal (1999), 18 (6), 1427-1434CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)
      A review with 55 refs. Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl-tRNA, either spontaneous dissocn. causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near-cognate tRNA; this error promotes a second frameshifting error (a dual-error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the spontaneous tRNA-induced frameshifting and programmed mRNA-induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome.
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    53. 53
      Sarr, M.; Kronqvist, N.; Chen, G.; Aleksis, R.; Purhonen, P.; Hebert, H.; Jaudzems, K.; Rising, A.; Johansson, J. A Spidroin-Derived Solubility Tag Enables Controlled Aggregation of a Designed Amyloid Protein. FEBS J. 2018, 285 (10), 1873– 1885,  DOI: 10.1111/febs.14451
      53
      A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein
      Sarr, Medoune; Kronqvist, Nina; Chen, Gefei; Aleksis, Rihards; Purhonen, Pasi; Hebert, Hans; Jaudzems, Kristaps; Rising, Anna; Johansson, Jan
      FEBS Journal (2018), 285 (10), 1873-1885CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Amyloidogenesis is assocd. with >30 diseases, but the mol. mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purifn., and exptl. studies challenging. NT* is a soly. tag derived from the N-terminal domain of spider silk protein fibroin that was recently introduced for the prodn. of several aggregation-prone peptides and proteins at high yields. Here, we investigate whether fusion to NT* could prevent amyloid fibril formation and enable controlled aggregation for exptl. studies. As an example of an amyloidogenic protein, we chose a de novo-designed polypeptide β17. Fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amts. of sol. and mostly monomeric protein. Structural anal. showed that β17 was kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopted a β-sheet conformation in a pH- and salt-dependent manner and assembled into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.
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    54. 54
      Vincenz-Donnelly, L.; Holthusen, H.; Körner, R.; Hansen, E. C.; Presto, J.; Johansson, J.; Sawarkar, R.; Hartl, F. U.; Hipp, M. S. High Capacity of the Endoplasmic Reticulum to Prevent Secretion and Aggregation of Amyloidogenic Proteins. EMBO J. 2018, 37 (3), 337– 350,  DOI: 10.15252/embj.201695841
      54
      High capacity of the endoplasmic reticulum to prevent secretion and aggregation of amyloidogenic proteins
      Vincenz-Donnelly, Lisa; Holthusen, Hauke; Koerner, Roman; Hansen, Erik C.; Presto, Jenny; Johansson, Jan; Sawarkar, Ritwick; Hartl, F. Ulrich; Hipp, Mark S.
      EMBO Journal (2018), 37 (3), 337-350CODEN: EMJODG; ISSN:0261-4189. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Protein aggregation is assocd. with neurodegeneration and various other pathologies. How specific cellular environments modulate the aggregation of disease proteins is not well understood. Here, we investigated how the endoplasmic reticulum (ER) quality control system handles β-sheet proteins that were designed de novo to form amyloid-like fibrils. While these proteins undergo toxic aggregation in the cytosol, we found that targeting them to the ER (ER-β) strongly reduced their toxicity. ER-β was retained within the ER in a sol., polymeric state, despite reaching very high concns. exceeding those of ER-resident mol. chaperones. ER-β was not removed by ER-assocd. degrdn. (ERAD), but interfered with the ERAD of other proteins. These findings demonstrate a remarkable capacity of the ER to prevent the formation of insol. β-aggregates and the secretion of potentially toxic protein species. These results also suggest a generic mechanism by which proteins with exposed β-sheet structure in the ER interfere with proteostasis.
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    55. 55
      Abelein, A.; Chen, G.; Kitoka, K.; Aleksis, R.; Oleskovs, F.; Sarr, M.; Landreh, M.; Pahnke, J.; Nordling, K.; Kronqvist, N.; Jaudzems, K.; Rising, A.; Johansson, J.; Biverstål, H. High-Yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain. Sci. Rep. 2020, 10 (1), 1– 10,  DOI: 10.1038/s41598-019-57143-x
      There is no corresponding record for this reference.
    56. 56
      Landreh, M.; Andersson, M.; Marklund, E. G.; Jia, Q.; Meng, Q.; Johansson, J.; Robinson, C. V.; Rising, A. Mass Spectrometry Captures Structural Intermediates in Protein Fiber Self-Assembly. Chem. Commun. 2017, 53 (23), 3319– 3322,  DOI: 10.1039/C7CC00307B
      56
      Mass spectrometry captures structural intermediates in protein fiber self-assembly
      Landreh, Michael; Andersson, Marlene; Marklund, Erik G.; Jia, Qiupin; Meng, Qing; Johansson, Jan; Robinson, Carol V.; Rising, Anna
      Chemical Communications (Cambridge, United Kingdom) (2017), 53 (23), 3319-3322CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Self-assembling proteins, the basis for a broad range of biol. scaffolds, are challenging to study using most structural biol. approaches. Here we show that mass spectrometry (MS) in combination with MD simulations captures structural features of short-lived oligomeric intermediates in spider silk formation, providing direct insights into its complex assembly process.
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    57. 57
      Jin, H. J.; Kaplan, D. L. Mechanism of Silk Processing in Insects and Spiders. Nature 2003, 424 (6952), 1057– 1061,  DOI: 10.1038/nature01809
      57
      Mechanism of silk processing in insects and spiders
      Jin, Hyoung-Joon; Kaplan, David L.
      Nature (London, United Kingdom) (2003), 424 (6952), 1057-1061CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      We report the identification of emulsion formation and micellar structures from aq. solns. of reconstituted silkworm silk fibroin as a first step in the process to control water and protein-protein interactions. The sizes (100-200 nm diam.) of these structures could be predicted from hydrophobicity plots of silk protein primary sequence. These micelles subsequently aggregated into larger globules' and gel-like states as the concn. of silk fibroin increased, while maintaining soly. owing to the hydrophilic regions of the protein interspersed among the larger hydrophobic regions. Upon phys. shearing or stretching structural transitions, increased birefringence and morphol. alignment were demonstrated, indicating that this process mimics the behavior of similar native silk proteins in vivo. Final morphol. features of these silk materials are similar to those obsd. in native silkworm fibers.
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    58. 58
      Schwarze, S.; Zwettler, F. U.; Johnson, C. M.; Neuweiler, H. The N-Terminal Domains of Spider Silk Proteins Assemble Ultrafast and Protected from Charge Screening. Nat. Commun. 2013, 4, 4,  DOI: 10.1038/ncomms3815
      There is no corresponding record for this reference.
    59. 59
      Ittah, S.; Cohen, S.; Garty, S.; Cohn, D.; Gat, U. An Essential Role for the C-Terminal Domain of a Dragline Spider Silk Protein in Directing Fiber Formation. Biomacromolecules 2006, 7 (6), 1790– 1795,  DOI: 10.1021/bm060120k
      59
      An Essential Role for the C-Terminal Domain of A Dragline Spider Silk Protein in Directing Fiber Formation
      Ittah, Shmulik; Cohen, Shulamit; Garty, Shai; Cohn, Daniel; Gat, Uri
      Biomacromolecules (2006), 7 (6), 1790-1795CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      We have employed baculovirus-mediated expression of the recombinant A. diadematus spider dragline silk fibroin rADF-4 to explore the role of the evolutionarily conserved C-terminal domain in self-assembly of the protein into fibers. In this unique system, polymn. of monomers occurs in the cytoplasm of living cells, giving rise to superfibers, which resemble some properties of the native dragline fibers that are synthesized by the spider using mech. spinning. While the C-terminal domain-contg. rADF-4 self-assembled to create intricate fibers in the host insect cells, a C-terminal deleted form of the protein (rADF-4-ΔC) self-assembled to create aggregates, which preserved the chem. stability of dragline fibers, yet lacked their shape. Interestingly, ultrastructural anal. showed that the rADF-4-ΔC monomers did form rudimentary nanofibers, but these were short and crude as compared to those of rADF-4, thus not supporting formation of the highly compact and oriented "superfiber" typical to the rADF-4 form. In addn., using thermal anal., we show evidence that the rADF-4 fibers but not the rADF-4-ΔC aggregates contain cryst. domains, further establishing the former as a veritable model of authentic dragline fibers. Thus, we conclude that the conserved C-terminal domain of dragline silk is important for the correct structure of the basic nanofibers, which assemble in an oriented fashion to form the final intricate natural-like dragline silk fiber.
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    60. 60
      Nguyen, P. Q.; Courchesne, N.-M. D.; Duraj-Thatte, A.; Praveschotinunt, P.; Joshi, N. S. Engineering Living Materials: Prospects and Challenges for Using Biological Systems to Direct the Assembly of Smart Materials. Adv. Mater. 2018, 30 (12), 139– 148,  DOI: 10.1002/adma.201704847
      There is no corresponding record for this reference.
    61. 61
      Chen, A. Y.; Deng, Z.; Billings, A. N.; Seker, U. O. S.; Lu, M. Y.; Citorik, R. J.; Zakeri, B.; Lu, T. K. Synthesis and Patterning of Tunable Multiscale Materials with Engineered Cells. Nat. Mater. 2014, 13 (5), 515– 523,  DOI: 10.1038/nmat3912
      61
      Synthesis and patterning of tunable multiscale materials with engineered cells
      Chen, Allen Y.; Deng, Zhengtao; Billings, Amanda N.; Seker, Urartu O. S.; Lu, Michelle Y.; Citorik, Robert J.; Zakeri, Bijan; Lu, Timothy K.
      Nature Materials (2014), 13 (5), 515-523CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
      Many natural biol. systems-such as biofilms, shells and skeletal tissues-are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid prodn., we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorg. materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based elec. switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.
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    62. 62
      Nguyen, P. Q.; Botyanszki, Z.; Tay, P. K. R.; Joshi, N. S. Programmable Biofilm-Based Materials from Engineered Curli Nanofibres. Nat. Commun. 2014, 5, 1– 10,  DOI: 10.1038/ncomms5945
      There is no corresponding record for this reference.
    63. 63
      Duraj-Thatte, A. M.; Manjula-Basavanna, A.; Courchesne, N. M. D.; Cannici, G. I.; Sánchez-Ferrer, A.; Frank, B. P.; van’t Hag, L.; Cotts, S. K.; Fairbrother, D. H.; Mezzenga, R.; Joshi, N. S. Water-Processable, Biodegradable and Coatable Aquaplastic from Engineered Biofilms. Nat. Chem. Biol. 2021, 17, 732,  DOI: 10.1038/s41589-021-00773-y
      63
      Water-processable, biodegradable and coatable aquaplastic from engineered biofilms
      Duraj-Thatte, Anna M.; Manjula-Basavanna, Avinash; Courchesne, Noemie-Manuelle Dorval; Cannici, Giorgia I.; Sanchez-Ferrer, Antoni; Frank, Benjamin P.; van't Hag, Leonie; Cotts, Sarah K.; Fairbrother, D. Howard; Mezzenga, Raffaele; Joshi, Neel S.
      Nature Chemical Biology (2021), 17 (6), 732-738CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
      Petrochem.-based plastics have not only contaminated all parts of the globe, but are also causing potentially irreversible damage to our ecosystem because of their non-biodegradability. As bioplastics are limited in no., there is an urgent need to design and develop more biodegradable alternatives to mitigate the plastic menace. In this regard, we report aquaplastic, a new class of microbial biofilm-based biodegradable bioplastic that is water-processable, robust, templatable and coatable. Here, Escherichia coli was genetically engineered to produce protein-based hydrogels, which are cast and dried under ambient conditions to produce aquaplastic, which can withstand strong acid/base and org. solvents. In addn., aquaplastic can be healed and welded to form three-dimensional architectures using water. The combination of straightforward microbial fabrication, water processability and biodegradability makes aquaplastic a unique material worthy of further exploration for packaging and coating applications.
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    64. 64
      Dubey, G.; Mequanint, K. Conjugation of Fibronectin onto Three-Dimensional Porous Scaffolds for Vascular Tissue Engineering Applications. Acta Biomater. 2011, 7 (3), 1114– 1125,  DOI: 10.1016/j.actbio.2010.11.010
      64
      Conjugation of fibronectin onto three-dimensional porous scaffolds for vascular tissue engineering applications
      Dubey, G.; Mequanint, K.
      Acta Biomaterialia (2011), 7 (3), 1114-1125CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
      Tissue engineering scaffolds provide the three-dimensional (3-D) geometry and mech. framework required for regulating cell behavior and facilitating tissue maturation. Unfortunately, most synthetic scaffolds lack the biol. recognition motifs required for seeded cell interaction. In order to impart this recognition, synthetic scaffolds should possess appropriate biol. functionality. Here, for the first time, we present a comprehensive study of fibronectin (FN) conjugation onto highly porous 3-D poly(carbonate) urethane scaffolds through grafted poly(acrylic acid) spacers on the urethane backbone. SEM was used to ensure that the porous structures of the scaffolds were preserved throughout the multiple conjugation steps, and Fourier transform IR spectroscopy was used to monitor the reaction progress. Toluidine blue staining revealed that increasing acrylic acid concn. and grafting time increased the no. of poly(acrylic acid) groups incorporated. High resoln. XPS studies of the scaffolds demonstrated an increase in nitrogen and sulfur due to FN conjugation. Immunofluorescence microscopy studies showed an even distribution of conjugated FN on the 3-D scaffolds. Cell culture studies using human coronary artery smooth muscle cells demonstrated that FN-conjugated scaffolds had improved cell attachment and infiltration depth compared with scaffolds without FN conjugation and with those scaffolds on which FN was merely adsorbed.
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    65. 65
      Glowacki, J.; Mizuno, S. Collagen Scaffolds for Tissue Engineering. Biopolymers 2008, 89 (5), 338– 344,  DOI: 10.1002/bip.20871
      65
      Collagen scaffolds for tissue engineering
      Glowacki, Julie; Mizuno, Shuichi
      Biopolymers (2008), 89 (5), 338-344CODEN: BIPMAA; ISSN:0006-3525. (John Wiley & Sons, Inc.)
      A review. There are two major approaches to tissue engineering for regeneration of tissues and organs. One involves cell-free materials and/or factors and one involves delivering cells to contribute to the regeneration process. Of the many scaffold materials being investigated, collagen type I, with selective removal of its telopeptides, has been shown to have many advantageous features for both of these approaches. Highly porous collagen lattice sponges have been used to support in vitro growth of many types of tissues. Use of bioreactors to control in vitro perfusion of medium and to apply hydrostatic fluid pressure has been shown to enhance histogenesis in collagen scaffolds. Collagen sponges have also been developed to contain differentiating-inducing materials like demineralized bone to stimulate differentiation of cartilage tissue both in vitro and in vivo.
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    66. 66
      Boccaccini, A. R.; Blaker, J. J. Bioactive Composite Materials for Tissue Engineering Scaffolds. Expert Rev. Med. Devices 2005, 2 (3), 303– 317,  DOI: 10.1586/17434440.2.3.303
      66
      Bioactive composite materials for tissue engineering scaffolds
      Boccaccini, Aldo R.; Blaker, Jonny J.
      Expert Review of Medical Devices (2005), 2 (3), 303-317CODEN: ERMDDX; ISSN:1743-4440. (Future Drugs Ltd.)
      A review. Synthetic bioactive and bioresorbable composite materials are becoming increasingly important as scaffolds for tissue engineering. Next-generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the scaffold by the regenerating tissue. Certain bioactive ceramics such as tricalcium phosphate and hydroxyapatite as well as bioactive glasses, such as 45S5 Bioglass, react with physiol. fluids to form tenacious bonds with hard (and in some cases soft) tissue. However, these bioactive materials are relatively stiff, brittle and difficult to form into complex shapes. Conversely, synthetic bioresorbable polymers are easily fabricated into complex structures, yet they are too weak to meet the demands of surgery and the in vivo physiol. environment. Composites of tailored phys., biol. and mech. properties as well as predictable degrdn. behavior can be produced combining bioresorbable polymers and bioactive inorg. phases. This review covers recent international research presenting the state-of-the-art development of these composite systems in terms of material constituents, fabrication technologies, structural and bioactive properties, as well as in vitro and in vivo characteristics for applications in tissue engineering and tissue regeneration. These materials may represent the effective optimal soln. for tailored tissue engineering scaffolds, making tissue engineering a realistic clin. alternative in the near future.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00574.

    • Additional methods, data, and figures including fluorescence levels, performances, OD600 values, max growth rate, structures, illustrations, Northern blot image, densitometry results, flow chart, and sequences (PDF)

    Accession Codes

    Plasmid Availability: pBAD-tdTEVDB_C-15A/G4C (the reporter plasmid following two rounds of directed evolution) is available on AddGene.


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