Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

'Deadman' and 'Passcode' microbial kill switches for bacterial containment

Nature Chemical Biology volume 12, pages 82–86 (2016)Cite this article

Subjects

Abstract

Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Deadman kill switch.
Figure 2: The fail-safe mechanism for Deadman circuit activation.
Figure 3: Passcode kill switch.
Figure 4: Long-term circuit stability.

Similar content being viewed by others

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

Protein Data Bank

References

  1. Moe-Behrens, G.H., Davis, R. & Haynes, K.A. Preparing synthetic biology for the world. Front. Microbiol. 4, 5 (2013).

    Article  Google Scholar 

  2. Bacchus, W., Aubel, D. & Fussenegger, M. Biomedically relevant circuit-design strategies in mammalian synthetic biology. Mol. Syst. Biol. 9, 691 (2013).

    Article  CAS  Google Scholar 

  3. Olson, E.J. & Tabor, J.J. Post-translational tools expand the scope of synthetic biology. Curr. Opin. Chem. Biol. 16, 300–306 (2012).

    Article  CAS  Google Scholar 

  4. Wright, O., Delmans, M., Stan, G.B. & Ellis, T. GeneGuard: a modular plasmid system designed for biosafety. ACS Synth. Biol. 4, 307–316 (2015).

    Article  CAS  Google Scholar 

  5. Chappell, J. et al. The centrality of RNA for engineering gene expression. Biotechnol. J. 8, 1379–1395 (2013).

    Article  CAS  Google Scholar 

  6. Cameron, D.E., Bashor, C.J. & Collins, J.J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).

    Article  CAS  Google Scholar 

  7. Wright, O., Stan, G.B. & Ellis, T. Building-in biosafety for synthetic biology. Microbiology 159, 1221–1235 (2013).

    Article  CAS  Google Scholar 

  8. Steidler, L. et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat. Biotechnol. 21, 785–789 (2003).

    Article  CAS  Google Scholar 

  9. Rovner, A.J. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

    Article  CAS  Google Scholar 

  10. Mandell, D.J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    Article  CAS  Google Scholar 

  11. Callura, J.M., Dwyer, D.J., Isaacs, F.J., Cantor, C.R. & Collins, J.J. Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proc. Natl. Acad. Sci. USA 107, 15898–15903 (2010).

    Article  CAS  Google Scholar 

  12. Contreras, A., Molin, S. & Ramos, J.L. Conditional-suicide containment system for bacteria which mineralize aromatics. Appl. Environ. Microbiol. 57, 1504–1508 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Cai, Y. et al. Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc. Natl. Acad. Sci. USA 112, 1803–1808 (2015).

    Article  CAS  Google Scholar 

  14. Gallagher, R.R., Patel, J.R., Interiano, A.L., Rovner, A.J. & Isaacs, F.J. Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Res. 43, 1945–1954 (2015).

    Article  CAS  Google Scholar 

  15. Voigt, C.A. Genetic parts to program bacteria. Curr. Opin. Biotechnol. 17, 548–557 (2006).

    Article  CAS  Google Scholar 

  16. Jensen, L.B., Ramos, J.L., Kaneva, Z. & Molin, S. A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene. Appl. Environ. Microbiol. 59, 3713–3717 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Litcofsky, K.D., Afeyan, R.B., Krom, R.J., Khalil, A.S. & Collins, J.J. Iterative plug-and-play methodology for constructing and modifying synthetic gene networks. Nat. Methods 9, 1077–1080 (2012).

    Article  CAS  Google Scholar 

  18. Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    Article  CAS  Google Scholar 

  19. Sadler, J.R., Sasmor, H. & Betz, J.L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc. Natl. Acad. Sci. USA 80, 6785–6789 (1983).

    Article  CAS  Google Scholar 

  20. Cameron, D.E. & Collins, J.J. Tunable protein degradation in bacteria. Nat. Biotechnol. 32, 1276–1281 (2014).

    Article  CAS  Google Scholar 

  21. Cheng, S.C., Kim, R., King, K., Kim, S.H. & Modrich, P. Isolation of gram quantities of EcoRI restriction and modification enzymes from an overproducing strain. J. Biol. Chem. 259, 11571–11575 (1984).

    CAS  PubMed  Google Scholar 

  22. Smith, A.B. & Maxwell, A. A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site. Nucleic Acids Res. 34, 4667–4676 (2006).

    Article  CAS  Google Scholar 

  23. Zhang, Y. et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol. Cell 12, 913–923 (2003).

    Article  CAS  Google Scholar 

  24. Salis, H.M. The ribosome binding site calculator. Methods Enzymol. 498, 19–42 (2011).

    Article  CAS  Google Scholar 

  25. Meinhardt, S. & Swint-Kruse, L. Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: bioinformatics-based predictions generate true positives and false negatives. Proteins 73, 941–957 (2008).

    Article  CAS  Google Scholar 

  26. Meinhardt, S. et al. Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression. Nucleic Acids Res. 40, 11139–11154 (2012).

    Article  CAS  Google Scholar 

  27. Shis, D.L., Hussain, F., Meinhardt, S., Swint-Kruse, L. & Bennett, M.R. Modular, multi-input transcriptional logic gating with orthogonal LacI/GalR family chimeras. ACS Synth. Biol. 3, 645–651 (2014).

    Article  CAS  Google Scholar 

  28. Sousa, A., Bourgard, C., Wahl, L.M. & Gordo, I. Rates of transposition in Escherichia coli. Biol. Lett. 9, 20130838 (2013).

    Article  Google Scholar 

  29. Cuthbertson, L. & Nodwell, J.R. The TetR family of regulators. Microbiol. Mol. Biol. Rev. 77, 440–475 (2013).

    Article  CAS  Google Scholar 

  30. Finn, R.D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014).

    Article  CAS  Google Scholar 

  31. Ramos, J.L. et al. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 69, 326–356 (2005).

    Article  CAS  Google Scholar 

  32. Cebolla, A., Vázquez, M.E. & Palomares, A.J. Expression vectors for the use of eukaryotic luciferases as bacterial markers with different colors of luminescence. Appl. Environ. Microbiol. 61, 660–668 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Mignon, C., Sodoyer, R. & Werle, B. Antibiotic-free selection in biotherapeutics: now and forever. Pathogens 4, 157–181 (2015).

    Article  CAS  Google Scholar 

  34. Csörgo, B., Fehér, T., Tímár, E., Blattner, F.R. & Pósfai, G. Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs. Microb. Cell Fact. 11, 11 (2012).

    Article  Google Scholar 

  35. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

  36. Bell, C.E. & Lewis, M. A closer view of the conformation of the Lac repressor bound to operator. Nat. Struct. Biol. 7, 209–214 (2000).

    Article  CAS  Google Scholar 

  37. Friedman, A.M., Fischmann, T.O. & Steitz, T.A. Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721–1727 (1995).

    Article  CAS  Google Scholar 

  38. Lewis, M. et al. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247–1254 (1996).

    Article  CAS  Google Scholar 

  39. Schumacher, M.A., Choi, K.Y., Lu, F., Zalkin, H. & Brennan, R.G. Mechanism of corepressor-mediated specific DNA binding by the purine repressor. Cell 83, 147–155 (1995).

    Article  CAS  Google Scholar 

  40. Glasfeld, A., Koehler, A.N., Schumacher, M.A. & Brennan, R.G. The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions. J. Mol. Biol. 291, 347–361 (1999).

    Article  CAS  Google Scholar 

  41. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  Google Scholar 

  42. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  43. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1982).

  44. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  45. Wild, J., Hradecna, Z. & Szybalski, W. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res. 12, 1434–1444 (2002).

    Article  CAS  Google Scholar 

  46. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Han for assistance with molecular cloning. This work was supported by funding from the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, Office of Naval Research MURI grant N000141110725, Air Force Office of Scientific Research grant FA9550-14-1-0060 and the Howard Hughes Medical Institute.

Author information

Author notes

  1. Clement T Y Chan, Jeong Wook Lee and D Ewen Cameron: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Clement T Y Chan, Jeong Wook Lee, D Ewen Cameron, Caleb J Bashor & James J Collins

  2. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Clement T Y Chan, Jeong Wook Lee, D Ewen Cameron, Caleb J Bashor & James J Collins

  3. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Clement T Y Chan, Jeong Wook Lee, D Ewen Cameron, Caleb J Bashor & James J Collins

  4. Harvard–MIT Program in Health Sciences and Technology, Cambridge, Massachusetts, USA

    James J Collins

  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    James J Collins

  6. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    James J Collins

Authors
  1. Clement T Y Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeong Wook Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D Ewen Cameron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Caleb J Bashor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James J Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.T.Y.C., J.W.L., D.E.C., C.J.B. and J.J.C. designed the study, analyzed data and wrote the paper. C.T.Y.C. and J.W.L. performed the experiments.

Corresponding author

Correspondence to James J Collins.

Ethics declarations

Competing interests

The authors have filed a provisional application with the US Patent and Trademark Office on this work.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–21 and Supplementary Table 1. (PDF 12918 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chan, C., Lee, J., Cameron, D. et al. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82–86 (2016). https://doi.org/10.1038/nchembio.1979

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1979

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology