Bacillus subtilis Biosensor Engineered To Assess Meat SpoilageClick to copy article linkArticle link copied!
- Alicja Daszczuk
- Yonathan Dessalegne
- Ismaêl Drenth
- Elbrich Hendriks
- Emeraldo Jo
- Tom van Lente
- Arjan Oldebesten
- Jonathon Parrish
- Wlada Poljakova
- Annisa A. Purwanto
- Renske van Raaphorst
- Mirjam Boonstra
- Auke van Heel
- Martijn Herber
- Sjoerd van der Meulen
- Jeroen Siebring
- Robin A. Sorg
- Matthias Heinemann
- Oscar P. Kuipers
- Jan-Willem Veening
Abstract
Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium Bacillus subtilis as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was PsboA, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for B. subtilis and cloned PsboA as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in B. subtilis in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage.
This publication is licensed for personal use by The American Chemical Society.
SPECIAL ISSUE
This article is part of the
Results and Discussion
Figure 1
Figure 1. (A) Schematic representation of the meat-biosensor BioBrick BBa_K816000. (B) Schematic representation of the sacA B. subtilis integration vector BBa_K818000. The sacA homology regions are indicated. The cat gene provides chloramphenicol resistance in B. subtilis and the bla gene confers ampicillin resistance in E. coli. (C) B. subtilis cells harboring the biosensor-construct were grown to midexponential growth phase in LB-medium, split in two cultures and the headspace of either fresh meat (kept on ice) or of spoiled meat (kept at room temperature) was flushed continuously through the shaking cultures. Fluorescence (arbitrary units) of 10 000 cells was determined by flow cytometry at timely intervals and a typical outcome of data from cells incubated for 5 h is shown.
Methods
DNA Techniques, Media and Growth Conditions
DNA Microarrays
Total Aerobic Microbial Count Assays (TAMC)
Construction of Plasmids and Strains
part/plasmid | BioBrick number | description | source |
---|---|---|---|
PsboA | BBa_K818100 | promoter induced by rotten meat volatiles | this study |
PsboA-amilGFP | BBa_K818600 | production of yellow pigment amilGFP induced by rotten meat volatiles | this study |
K818000 | BBa_K818000 | plasmid replicates in E. coli and integrates into the Bacillus subtilis genome via double crossover; contains BioBrick MCS and double terminator B0015 | this study |
amilGFP | BBa_K592010 | fluorescent and bright colored protein | iGEM Uppsala 2011 |
K818000-amilGFP | intermediate plasmid for creation of PsboA-amilGFP in K818000 | this study | |
B0015 | BBa_B0015 | double terminator | parts registry |
RBS | BBa_B0034 | ribosome binding site used widely in the iGEM competition | parts registry |
Flow Cytometry
Supporting Information
Movie S1, a plasmid map and the sequence of the biosensor construct. Movie S1 shows the experimental setup: the headspace of trays containing either fresh meat (on ice) or spoiled meat (>106 CFU/g meat) (room temperature) are pumped through shaking cultures of the B. subtilis biosensor. This material is available free of charge via the Internet at http://pubs.acs.org.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
We thank the sponsors of the Groningen 2012 iGEM team (http://2012.igem.org/Team:Groningen/our_sponsors). We thank Roel Bovenberg, Gert-Jan Euverink, and Marnix Medema for valuable discussions.
References
This article references 11 other publications.
- 1Michener, J. K., Thodey, K., Liang, J. C., and Smolke, C. D. (2012) Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways Metab. Eng. 14, 212– 222Google Scholar1Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathwaysMichener, Joshua K.; Thodey, Kate; Liang, Joe C.; Smolke, Christina D.Metabolic Engineering (2012), 14 (3), 212-222CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)A review. Cells are filled with biosensors, mol. systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chem. reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metab. have focused on introducing new pathways and removing existing pathway regulation. Synthetic biol. is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biol. systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of mol. tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.
- 2van der Meer, J. R. and Belkin, S. (2010) Where microbiology meets microengineering: Design and applications of reporter bacteria Nat. Rev. Microbiol. 8, 511– 522Google Scholar2Where microbiology meets microengineering: design and applications of reporter bacteriavan der Meer, Jan Roelof; Belkin, ShimshonNature Reviews Microbiology (2010), 8 (7), 511-522CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)Bacteria have long been the targets for genetic manipulation, but more recently they have been synthetically designed to carry out specific tasks. Among the simplest of these tasks is chem. compd. and toxicity detection coupled to the prodn. of a quantifiable reporter signal. In this Review, we describe the current design of bacterial bioreporters and their use in a range of assays to measure the presence of harmful chems. in water, air, soil, food or biol. specimens. New trends for integrating synthetic biol. and microengineering into the design of bacterial bioreporter platforms are also highlighted.
- 3Weber, W., Luzi, S., Karlsson, M., and Fussenegger, M. (2009) A novel hybrid dual-channel catalytic-biological sensor system for assessment of fruit quality J. Biotechnol. 139, 314– 317Google Scholar3A novel hybrid dual-channel catalytic-biological sensor system for assessment of fruit qualityWeber, Wilfried; Luzi, Stefan; Karlsson, Maria; Fussenegger, MartinJournal of Biotechnology (2009), 139 (4), 314-317CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)The release of volatile ethylene and acetaldehyde characterizes the metabolic state and quality of fruit. We have designed and implemented a hybrid dual-channel catalytic-biol. sensor system, which is able to quantify both volatiles in situ. This sensor system consists of a mammalian cell line engineered for constitutive expression of an Aspergillus nidulans-derived biosensor which triggers quant. reporter gene expression in the presence of volatile acetaldehyde. Ethylene, oxidized to acetaldehyde using a Wacker-based process, can be quantified by the same transgenic sensor cell line. Differential profiling of reporter gene transcription by the sensor system revealed the relative concns. of both volatile metabolites and enabled correct assessment of fruit quality as shown for fresh, old and rotten apples. Functional combination of catalytic processes with biosensor technol. is able to precisely capture the metabolic state of food and may foster novel insight into biochem. food quality assessment as well as the design of synthetic control circuits detecting and preventing food spoilage.
- 4Kerry, J. P., O’Grady, M. N., and Hogan, S. A. (2006) Past, current, and potential utilisation of active and intelligent packaging systems for meat and muscle-based products: A review Meat Science 74, 113– 130Google ScholarThere is no corresponding record for this reference.
- 5Urban, A., Eckermann, S., Fast, B., Metzger, S., Gehling, M., Ziegelbauer, K., Rübsamen-Waigmann, H., and Freiberg, C. (2007) Novel whole-cell antibiotic biosensors for compound discovery Appl. Environ. Microbiol. 73, 6436– 6443Google ScholarThere is no corresponding record for this reference.
- 6Knecht, L. D., Pasini, P., and Daunert, S. (2011) Bacterial spores as platforms for bioanalytical and biomedical applications Anal. Bioanal. Chem. 400, 977– 989Google Scholar6Bacterial spores as platforms for bioanalytical and biomedical applicationsKnecht, Leslie D.; Pasini, Patrizia; Daunert, SylviaAnalytical and Bioanalytical Chemistry (2011), 400 (4), 977-989CODEN: ABCNBP; ISSN:1618-2642. (Springer)A review. Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, esp. under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An addnl. application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic prodn. of chems., and the delivery of biomols. of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanal. and biotechnol. applications, and the use of spores as vehicles for vaccines and therapeutic agents. FigureBacterial spores have been utilized in biotechnol. applications due to their innate stability under normal and extreme environmental conditions. Specifically, the spores have been employed to preserve whole-cell sensing systems (top panel) and to surface display heterologous proteins (bottom panel) in bioanal. and biomedical applications.
- 7Baerends, R. J., Smits, W. K., de Jong, A., Hamoen, L. W., Kok, J., and Kuipers, O. P. (2004) Genome2D: A visualization tool for the rapid analysis of bacterial transcriptome data Genome Biol. 5, R37Google ScholarThere is no corresponding record for this reference.
- 8Zheng, G., Yan, L. Z., Vederas, J. C., and Zuber, P. (1999) Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin J. Bacteriol. 181, 7346– 7355Google Scholar8Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosinZheng, Guolu; Yan, Liang Z.; Vederas, John C.; Zuber, PeterJournal of Bacteriology (1999), 181 (23), 7346-7355CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Bacillus subtilis JH642 and a wild strain of B. subtilis called 22a both produce an antilisterial peptide that can be purified by anion-exchange and gel filtration chromatog. Amino acid anal. confirmed that the substance was the cyclic bacteriocin subtilosin. A mutant defective in prodn. of the substance was isolated from a plasmid gene disruption library. The plasmid insertion conferring the antilisterial-peptide-neg. phenotype was located in a seven-gene operon (alb, for antilisterial bacteriocin) residing immediately downstream from the sbo gene, which encodes the precursor of subtilosin. An insertion mutation in the sbo gene also conferred loss of antilisterial activity. Comparison of the presubtilosin and mature subtilosin sequences suggested that certain residues undergo unusual posttranslational modifications unlike those occurring during the synthesis of class I (lantibiotic) or some class II bacteriocins. The putative products of the genes of the operon identified show similarities to peptidases and transport proteins that may function in processing and export. Two alb gene products resemble proteins that function in pyrroloquinoline quinone biosynthesis. The use of lacZ-alb and lacZ-sbo gene fusions, along with primer extension anal., revealed that the sbo-alb genes are transcribed from a major promoter, residing upstream of sbo, that is very likely utilized by the σA form of RNA polymerase. The sbo and alb genes are neg. regulated by the global transition state regulator AbrB and are also under pos. autoregulation that is not mediated by the subtilosin peptide but instead requires one or more of the alb gene products.
- 9Middleton, R. and Hofmeister, A. (2004) New shuttle vectors for ectopic insertion of genes into Bacillus subtilis Plasmid 51, 238– 245Google ScholarThere is no corresponding record for this reference.
- 10Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., Miller, D. J., Wiedenmann, J., Salih, A., and Matz, M. V. (2008) Diversity and evolution of coral fluorescent proteins PLoS One 3, e2680Google ScholarThere is no corresponding record for this reference.
- 11Harwood, C. R. and Cutting, S. M. (1990) Molecular Biological Methods for Bacillus, John Wiley and Sons Ltd., Chichester.Google ScholarThere is no corresponding record for this reference.
Cited By
Smart citations by scite.ai include citation statements extracted from the full text of the citing article. The number of the statements may be higher than the number of citations provided by ACS Publications if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
This article is cited by 17 publications.
- Dengbin Yu, Rongbing Li, Xiaoxuan Sun, He Zhang, Hongwen Yu, Shaojun Dong. Colorimetric and Electrochemical Dual-Signal Method for Water Toxicity Detection Based on Escherichia coli and p-Benzoquinone. ACS Sensors 2021, 6
(7)
, 2674-2681. https://doi.org/10.1021/acssensors.1c00651
- Valeriy Zaytsev, Maria N. Tutukina, Margarita R. Chetyrkina, Pavel V. Shelyakin, George Ovchinnikov, Dina Satybaldina, Vladislav A. Kondrashov, Maria S. Bandurist, Shakhmaran Seilov, Dmitry A. Gorin, Fedor S. Fedorov, Mikhail S. Gelfand, Albert G. Nasibulin. Monitoring of meat quality and change-point detection by a sensor array and profiling of bacterial communities. Analytica Chimica Acta 2024, 1320 , 343022. https://doi.org/10.1016/j.aca.2024.343022
- Carlos Alberto Guerra, Lucas Marques Costa, Vanessa Sales de Oliveira, Breno Pereira de Paula, Wilson José Fernandes Lemos Junior, Rosa Helena Luchese, Viviana Corich, Alessio Giacomini, André Fioravante Guerra. Correlation between natural microbial load and formation of ropy slime affecting the superficial color of vacuum-packaged cooked sausage. Meat Science 2023, 201 , 109197. https://doi.org/10.1016/j.meatsci.2023.109197
- Subhadeep Mandal, Ganesh Chandra Banik. Biosensors for Precision Agriculture. 2023, 709-727. https://doi.org/10.1016/B978-0-12-822548-6.00150-3
- Shuqi Li, Yu Li, Jingkun Li, Jinghan Liu, Fuwei Pi, Jianfeng Ping. Recent advances of three-dimensional micro-environmental constructions on cell-based biosensors and perspectives in food safety. Biosensors and Bioelectronics 2022, 216 , 114601. https://doi.org/10.1016/j.bios.2022.114601
- Pramod Kumar Nanda, Dipanwita Bhattacharya, Jyotishka Kumar Das, Samiran Bandyopadhyay, Daniel Ekhlas, Jose M. Lorenzo, Premanshu Dandapat, Laura Alessandroni, Arun K. Das, Mohammed Gagaoua. Emerging Role of Biosensors and Chemical Indicators to Monitor the Quality and Safety of Meat and Meat Products. Chemosensors 2022, 10
(8)
, 322. https://doi.org/10.3390/chemosensors10080322
- Mukunda Goswami, Yashwanth Belathur Shambhugowda, Arjunan Sathiyanarayanan, Nevil Pinto, Alexandrea Duscher, Reza Ovissipour, Wazir Singh Lakra, Ravishankar Chandragiri Nagarajarao. Cellular Aquaculture: Prospects and Challenges. Micromachines 2022, 13
(6)
, 828. https://doi.org/10.3390/mi13060828
- Junaid Siddiqui, Mahtab Taheri, Arif Ul Alam, M. Jamal Deen. Nanomaterials in Smart Packaging Applications: A Review. Small 2022, 18
(1)
https://doi.org/10.1002/smll.202101171
- Barbara Sionek, Wiesław Przybylski, Krzysztof Tambor. Biosensors in Evaluation of Quality of Meat and Meat Products – A Review. Annals of Animal Science 2020, 20
(4)
, 1151-1168. https://doi.org/10.2478/aoas-2020-0057
- Josefine Liljeruhm, Saskia K. Funk, Sandra Tietscher, Anders D. Edlund, Sabri Jamal, Pikkei Wistrand-Yuen, Karl Dyrhage, Arvid Gynnå, Katarina Ivermark, Jessica Lövgren, Viktor Törnblom, Anders Virtanen, Erik R. Lundin, Erik Wistrand-Yuen, Anthony C. Forster. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 2018, 12
(1)
https://doi.org/10.1186/s13036-018-0100-0
- , , Meixi Ling, Jianghua Li, Guocheng Du, Long Liu. Metabolic engineering for the production of chitooligosaccharides: advances and perspectives. Emerging Topics in Life Sciences 2018, 2
(3)
, 377-388. https://doi.org/10.1042/ETLS20180009
- Wenjing Cui, Laichuang Han, Feiya Suo, Zhongmei Liu, Li Zhou, Zhemin Zhou. Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond. World Journal of Microbiology and Biotechnology 2018, 34
(10)
https://doi.org/10.1007/s11274-018-2531-7
- Clémence Roggo, Cristian Picioreanu, Xavier Richard, Christian Mazza, Harald van Lintel, Jan Roelof van der Meer. Quantitative chemical biosensing by bacterial chemotaxis in microfluidic chips. Environmental Microbiology 2018, 20
(1)
, 241-258. https://doi.org/10.1111/1462-2920.13982
- Preetam Anbukarasu, Dominic Sauvageau, Anastasia L. Elias. Time‐Temperature Indicator Based on Enzymatic Degradation of Dye‐Loaded Polyhydroxybutyrate. Biotechnology Journal 2017, 12
(9)
https://doi.org/10.1002/biot.201700050
- Clémence Roggo, Jan Roelof van der Meer. Miniaturized and integrated whole cell living bacterial sensors in field applicable autonomous devices. Current Opinion in Biotechnology 2017, 45 , 24-33. https://doi.org/10.1016/j.copbio.2016.11.023
- Sen Yang, Guocheng Du, Jian Chen, Zhen Kang. Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Applied Microbiology and Biotechnology 2017, 101
(10)
, 4151-4161. https://doi.org/10.1007/s00253-017-8142-7
- Richard Kelwick, Laura Bowater, Kay H. Yeoman, Richard P. Bowater, . Promoting microbiology education through the iGEM synthetic biology competition. FEMS Microbiology Letters 2015, 362
(16)
, fnv129. https://doi.org/10.1093/femsle/fnv129
Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.
Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.
The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.
Recommended Articles
Abstract
Figure 1
Figure 1. (A) Schematic representation of the meat-biosensor BioBrick BBa_K816000. (B) Schematic representation of the sacA B. subtilis integration vector BBa_K818000. The sacA homology regions are indicated. The cat gene provides chloramphenicol resistance in B. subtilis and the bla gene confers ampicillin resistance in E. coli. (C) B. subtilis cells harboring the biosensor-construct were grown to midexponential growth phase in LB-medium, split in two cultures and the headspace of either fresh meat (kept on ice) or of spoiled meat (kept at room temperature) was flushed continuously through the shaking cultures. Fluorescence (arbitrary units) of 10 000 cells was determined by flow cytometry at timely intervals and a typical outcome of data from cells incubated for 5 h is shown.
References
This article references 11 other publications.
- 1Michener, J. K., Thodey, K., Liang, J. C., and Smolke, C. D. (2012) Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways Metab. Eng. 14, 212– 2221Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathwaysMichener, Joshua K.; Thodey, Kate; Liang, Joe C.; Smolke, Christina D.Metabolic Engineering (2012), 14 (3), 212-222CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)A review. Cells are filled with biosensors, mol. systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chem. reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metab. have focused on introducing new pathways and removing existing pathway regulation. Synthetic biol. is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biol. systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of mol. tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.
- 2van der Meer, J. R. and Belkin, S. (2010) Where microbiology meets microengineering: Design and applications of reporter bacteria Nat. Rev. Microbiol. 8, 511– 5222Where microbiology meets microengineering: design and applications of reporter bacteriavan der Meer, Jan Roelof; Belkin, ShimshonNature Reviews Microbiology (2010), 8 (7), 511-522CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)Bacteria have long been the targets for genetic manipulation, but more recently they have been synthetically designed to carry out specific tasks. Among the simplest of these tasks is chem. compd. and toxicity detection coupled to the prodn. of a quantifiable reporter signal. In this Review, we describe the current design of bacterial bioreporters and their use in a range of assays to measure the presence of harmful chems. in water, air, soil, food or biol. specimens. New trends for integrating synthetic biol. and microengineering into the design of bacterial bioreporter platforms are also highlighted.
- 3Weber, W., Luzi, S., Karlsson, M., and Fussenegger, M. (2009) A novel hybrid dual-channel catalytic-biological sensor system for assessment of fruit quality J. Biotechnol. 139, 314– 3173A novel hybrid dual-channel catalytic-biological sensor system for assessment of fruit qualityWeber, Wilfried; Luzi, Stefan; Karlsson, Maria; Fussenegger, MartinJournal of Biotechnology (2009), 139 (4), 314-317CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)The release of volatile ethylene and acetaldehyde characterizes the metabolic state and quality of fruit. We have designed and implemented a hybrid dual-channel catalytic-biol. sensor system, which is able to quantify both volatiles in situ. This sensor system consists of a mammalian cell line engineered for constitutive expression of an Aspergillus nidulans-derived biosensor which triggers quant. reporter gene expression in the presence of volatile acetaldehyde. Ethylene, oxidized to acetaldehyde using a Wacker-based process, can be quantified by the same transgenic sensor cell line. Differential profiling of reporter gene transcription by the sensor system revealed the relative concns. of both volatile metabolites and enabled correct assessment of fruit quality as shown for fresh, old and rotten apples. Functional combination of catalytic processes with biosensor technol. is able to precisely capture the metabolic state of food and may foster novel insight into biochem. food quality assessment as well as the design of synthetic control circuits detecting and preventing food spoilage.
- 4Kerry, J. P., O’Grady, M. N., and Hogan, S. A. (2006) Past, current, and potential utilisation of active and intelligent packaging systems for meat and muscle-based products: A review Meat Science 74, 113– 130There is no corresponding record for this reference.
- 5Urban, A., Eckermann, S., Fast, B., Metzger, S., Gehling, M., Ziegelbauer, K., Rübsamen-Waigmann, H., and Freiberg, C. (2007) Novel whole-cell antibiotic biosensors for compound discovery Appl. Environ. Microbiol. 73, 6436– 6443There is no corresponding record for this reference.
- 6Knecht, L. D., Pasini, P., and Daunert, S. (2011) Bacterial spores as platforms for bioanalytical and biomedical applications Anal. Bioanal. Chem. 400, 977– 9896Bacterial spores as platforms for bioanalytical and biomedical applicationsKnecht, Leslie D.; Pasini, Patrizia; Daunert, SylviaAnalytical and Bioanalytical Chemistry (2011), 400 (4), 977-989CODEN: ABCNBP; ISSN:1618-2642. (Springer)A review. Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, esp. under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An addnl. application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic prodn. of chems., and the delivery of biomols. of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanal. and biotechnol. applications, and the use of spores as vehicles for vaccines and therapeutic agents. FigureBacterial spores have been utilized in biotechnol. applications due to their innate stability under normal and extreme environmental conditions. Specifically, the spores have been employed to preserve whole-cell sensing systems (top panel) and to surface display heterologous proteins (bottom panel) in bioanal. and biomedical applications.
- 7Baerends, R. J., Smits, W. K., de Jong, A., Hamoen, L. W., Kok, J., and Kuipers, O. P. (2004) Genome2D: A visualization tool for the rapid analysis of bacterial transcriptome data Genome Biol. 5, R37There is no corresponding record for this reference.
- 8Zheng, G., Yan, L. Z., Vederas, J. C., and Zuber, P. (1999) Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin J. Bacteriol. 181, 7346– 73558Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosinZheng, Guolu; Yan, Liang Z.; Vederas, John C.; Zuber, PeterJournal of Bacteriology (1999), 181 (23), 7346-7355CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Bacillus subtilis JH642 and a wild strain of B. subtilis called 22a both produce an antilisterial peptide that can be purified by anion-exchange and gel filtration chromatog. Amino acid anal. confirmed that the substance was the cyclic bacteriocin subtilosin. A mutant defective in prodn. of the substance was isolated from a plasmid gene disruption library. The plasmid insertion conferring the antilisterial-peptide-neg. phenotype was located in a seven-gene operon (alb, for antilisterial bacteriocin) residing immediately downstream from the sbo gene, which encodes the precursor of subtilosin. An insertion mutation in the sbo gene also conferred loss of antilisterial activity. Comparison of the presubtilosin and mature subtilosin sequences suggested that certain residues undergo unusual posttranslational modifications unlike those occurring during the synthesis of class I (lantibiotic) or some class II bacteriocins. The putative products of the genes of the operon identified show similarities to peptidases and transport proteins that may function in processing and export. Two alb gene products resemble proteins that function in pyrroloquinoline quinone biosynthesis. The use of lacZ-alb and lacZ-sbo gene fusions, along with primer extension anal., revealed that the sbo-alb genes are transcribed from a major promoter, residing upstream of sbo, that is very likely utilized by the σA form of RNA polymerase. The sbo and alb genes are neg. regulated by the global transition state regulator AbrB and are also under pos. autoregulation that is not mediated by the subtilosin peptide but instead requires one or more of the alb gene products.
- 9Middleton, R. and Hofmeister, A. (2004) New shuttle vectors for ectopic insertion of genes into Bacillus subtilis Plasmid 51, 238– 245There is no corresponding record for this reference.
- 10Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., Miller, D. J., Wiedenmann, J., Salih, A., and Matz, M. V. (2008) Diversity and evolution of coral fluorescent proteins PLoS One 3, e2680There is no corresponding record for this reference.
- 11Harwood, C. R. and Cutting, S. M. (1990) Molecular Biological Methods for Bacillus, John Wiley and Sons Ltd., Chichester.There is no corresponding record for this reference.
Supporting Information
Supporting Information
Movie S1, a plasmid map and the sequence of the biosensor construct. Movie S1 shows the experimental setup: the headspace of trays containing either fresh meat (on ice) or spoiled meat (>106 CFU/g meat) (room temperature) are pumped through shaking cultures of the B. subtilis biosensor. This material is available free of charge via the Internet at http://pubs.acs.org.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.