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Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron

An Author Correction to this article was published on 07 May 2020

This article has been updated


Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome1. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron2,3,4,5. We report implementation of Cello circuit design automation software6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR–dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.

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Fig. 1: Sensor, NOT and NOR gate characterization in B. thetaiotaomicron.
Fig. 2: Automated design of an XOR circuit.
Fig. 3: A circuit designed to integrate two sensors and control three programs of gene expression.
Fig. 4: Coculture of B. thetaiotaomicron with colonic epithelial monolayer.

Data availability

Genetic parts and the UCF file Bth1C1G1T1 are available as Supplementary Information. The DNA sequences for the following plasmids are deposited into GenBank: pMT405 (MN991273); pMT406 (MN991274); pMT423 (MN991275); pMT444 (MN991276); pMT445 (MN991277); pMT447 (MN991278); pMT448 (MN991279); pMT449 (MN991280); pMT450 (MN991281); pMT451 (MN991282); pMT455 (MN991283); pMT462 (MN991284); pMT468 (MN991285); pMT469 (MN991286); pMT470 (MN991287); pMT492 (MN991288); pMT493 (MN991289); pMT494 (MN991290).

Code availability

The Cello software and codes are freely available (

Change history

  • 07 May 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


  1. 1.

    Claesen, J. & Fischbach, M. A. Synthetic microbes as drug delivery systems. ACS Synth. Biol. 4, 358–364 (2015).

    CAS  PubMed  Google Scholar 

  2. 2.

    Mimee, M., Tucker, A. C., Voigt, C. A. & Lu, T. K. Programming a human commensal bacterium, bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Syst. 1, 62–71 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Lim, B., Zimmermann, M., Barry, N. A. & Goodman, A. L. Engineered regulatory systems modulate gene expression of human commensals in the gut. Cell 169, 547–558.e515 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Whitaker, W. R., Shepherd, E. S. & Sonnenburg, J. L. Tunable expression tools enable single-cell strain distinction in the gut microbiome. Cell 169, 538–546.e512 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Ruder, W. C., Lu, T. & Collins, J. J. Synthetic biology moving into the clinic. Science 333, 1248–1252 (2011).

    CAS  PubMed  Google Scholar 

  6. 6.

    Nielsen, A. A. et al. Genetic circuit design automation. Science 352, aac7341 (2016).

    PubMed  Google Scholar 

  7. 7.

    Ozdemir, T., Fedorec, A. J. H., Danino, T. & Barnes, C. P. Synthetic biology and engineered live biotherapeutics: toward increasing system complexity. Cell Syst. 7, 5–16 (2018).

    CAS  PubMed  Google Scholar 

  8. 8.

    Lee, J. W., Chan, C. T. Y., Slomovic, S. & Collins, J. J. Next-generation biocontainment systems for engineered organisms. Nat. Chem. Biol. 14, 530–537 (2018).

    CAS  PubMed  Google Scholar 

  9. 9.

    Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A. & Shapiro, M. G. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat. Chem. Biol. 13, 75–80 (2017).

    CAS  PubMed  Google Scholar 

  10. 10.

    Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Riglar, D. T. & Silver, P. A. Engineering bacteria for diagnostic and therapeutic applications. Nat. Rev. Microbiol. 16, 214–225 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Wexler, A. G. & Goodman, A. L. An insider’s perspective: Bacteroides as a window into the microbiome. Nat. Microbiol. 2, 17026 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Schwalm, N. D. & Groisman, E. A. Navigating the gut buffet: Control of polysaccharide utilization in bacteroides spp. Trends Microbiol. 25, 1005–1015 (2017).

    CAS  PubMed  Google Scholar 

  14. 14.

    Faith, J. J. et al. The long-term stability of the human gut microbiota. Science 341, 1237439 (2013).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Donaldson, G. P., Lee, S. M. & Mazmanian, S. K. Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 14, 20–32 (2015).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Jacobson, A. et al. A gut commensal-produced metabolite mediates colonization resistance to salmonella infection. Cell Host Microbe 24, 296–307.e297 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Russell, AlistairB. et al. A type VI secretion-related pathway in bacteroidetes mediates interbacterial antagonism. Cell Host Microbe 16, 227–236 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ramakrishna, C. et al. Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis. Nat. Commun. 10, 2153 (2019).

  19. 19.

    Wegorzewska, M. M. et al. Diet modulates colonic T cell responses by regulating the expression of a Bacteroides thetaiotaomicron antigen. Science Immunol. 4, eaau9079 (2019).

    Google Scholar 

  20. 20.

    Hamady, Z. Z. R. et al. Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus. Gut 59, 461–469 (2010).

    CAS  PubMed  Google Scholar 

  21. 21.

    Round, J. L. & Mazmanian, S. K. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. PNAS 107, 12204–12209 (2010).

    CAS  PubMed  Google Scholar 

  22. 22.

    Shepherd, E. S., DeLoache, W. C., Pruss, K. M., Whitaker, W. R. & Sonnenburg, J. L. An exclusive metabolic niche enables strain engraftment in the gut microbiota. Nature 557, 434–438 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Qi, LeiS. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Daeffler, K. N. M. et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol. Syst. Biol. 13, 923 (2017).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Riglar, D. T. et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat. Biotechnol. 35, 653–658 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Archer, E. J., Robinson, A. B. & Süel, G. M. Engineered E. coli that detect and respond to gut inflammation through nitric oxide sensing. ACS Synth. Biol. 1, 451–457 (2012).

    CAS  PubMed  Google Scholar 

  27. 27.

    Hwang, I. Y. et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8, 1–11 (2017).

    CAS  Google Scholar 

  28. 28.

    Chen, Y. et al. Robust bioengineered 3D functional human intestinal epithelium. Sci. Rep. 5, 1–11 (2015).

    Google Scholar 

  29. 29.

    Leschner, S. et al. Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic. Nucleic Acids Res. 40, 2984–2994 (2012).

    CAS  PubMed  Google Scholar 

  30. 30.

    Ozdemir, T. Design and construction of therapeutic bacterial sensors in Escherichia coli Nissle 1917. PhD thesis, University College London (2018).

  31. 31.

    Kotula, J. W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. PNAS 111, 4838–4843 (2014).

    CAS  PubMed  Google Scholar 

  32. 32.

    Kim, S. et al. Quorum sensing can be repurposed to promote information transfer between bacteria in the mammalian gut. ACS Synth. Biol. 7, 2270–2281 (2018).

    CAS  PubMed  Google Scholar 

  33. 33.

    Chen, J. X. et al. Development of aspirin-inducible biosensors in Escherichia coli and SimCells. Appl. Environ. Microbiol. 85, e02959-18 (2019).

  34. 34.

    Mimee, M., Citorik, R. J. & Lu, T. K. Microbiome therapeutics - Advances and challenges. Adv. Drug Deliv. Rev. 105, 44–54 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Yao, L. et al. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 7, e37182 (2018).

  36. 36.

    Ridlon, J. M., Kang, D. J., Hylemon, P. B. & Bajaj, J. S. Bile acids and the gut microbiome. Curr. Opin. Gastroenterol. 30, 332–338 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Jason M. Ridlon, S. C. H. S. B. D.-J. K., Phillip & Hylemon, B. Consequences of bile salt biotransformations by intestinal bacteria. Gut Microbes 7, 22–39 (2016).

  38. 38.

    Degirolamo, C., Modica, S., Palasciano, G. & Moschetta, A. Bile acids and colon cancer: Solving the puzzle with nuclear receptors. Trends Mol. Med. 17, 564–572 (2011).

    CAS  PubMed  Google Scholar 

  39. 39.

    Kawamata, Y. et al. A G protein-coupled receptor responsive to bile acids. J. Biol. Chem. 278, 9435–9440 (2003).

    CAS  PubMed  Google Scholar 

  40. 40.

    Hofmann, A. F. & Hagey, L. R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell. Mol. Life Sci. 65, 2461–2483 (2008).

    CAS  PubMed  Google Scholar 

  41. 41.

    Hamilton, J. P. et al. Human cecal bile acids: concentration and spectrum. Am. J. Physiol. 293, G256–G263 (2007).

    CAS  Google Scholar 

  42. 42.

    Li, T. & Chiang, J. Y. L. Bile acids as metabolic regulators. Curr. Opin. Gastroenterol. 31, 159–165 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Ung, K. A., Gillberg, R., Kilander, A. & Abrahamsson, H. Role of bile acids and bile acid binding agents in patients with collagenous colitis. Gut 46, 170–175 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Bernstein, H., Bernstein, C., Payne, C. M., Dvorakova, K. & Garewal, H. Bile acids as carcinogens in human gastrointestinal cancers. Mutat. Res. 589, 47–65 (2005).

    CAS  PubMed  Google Scholar 

  45. 45.

    Cerda-Maira, F. A., Ringelberg, C. S. & Taylor, R. K. The bile response repressor BreR regulates expression of the vibrio cholerae breAB efflux system operon. J. Bacteriol. 190, 7441–7452 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Gunn, J. S. Mechanisms of bacterial resistance and response to bile. Microbes Infect. 2, 907–913 (2000).

    CAS  PubMed  Google Scholar 

  47. 47.

    Lin, J. et al. Bile salts modulate expression of the CmeABC multidrug efflux pump in campylobacter jejuni. J. Bacteriol. 187, 7417–7424 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Rössger, K., Charpin-El-Hamri, G. & Fussenegger, M. Bile acid-controlled transgene expression in mammalian cells and mice. Metab. Eng. 21, 81–90 (2014).

    PubMed  Google Scholar 

  49. 49.

    Din, M. O. et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81–85 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Riglar, D. T. et al. Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator. Nat. Commun. 10, 1–12 (2019).

    CAS  Google Scholar 

  51. 51.

    Oberortner, E., Bhatia, S., Lindgren, E. & Densmore, D. A rule-based design specification language for synthetic biology. J. Emerg. Technol. Comput. Syst. 11, 25:21–25:19 (2014).

    Google Scholar 

  52. 52.

    Kelly, J. R. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J. Biol. Eng. 3, 4 (2009).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Cuthbertson, L., Ahn, SangK. & Nodwell, JustinR. Deglycosylation as a mechanism of inducible antibiotic resistance revealed using a global relational tree for one-component regulators. Chem. Biol. 20, 232–240 (2013).

    CAS  PubMed  Google Scholar 

  55. 55.

    Cerda-Maira, F. A., Kovacikova, G., Jude, B. A., Skorupski, K. & Taylor, R. K. Characterization of BreR interaction with the bile response promoters breAB and breR in Vibrio cholerae. J. Bacteriol. 195, 307–317 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Stanton, B. C. et al. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat. Chem. Biol. 10, 99–105 (2014).

    CAS  PubMed  Google Scholar 

  57. 57.

    Nielsen, A. A. K. & Voigt, C. A. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol. Syst. Biol. 10, n/a–n/a (2014).

    Google Scholar 

  58. 58.

    Zhang, S. & Voigt, C. A. Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design. Nucl. Acids Res. 46, 11115–11125 (2018).

    CAS  PubMed  Google Scholar 

  59. 59.

    Gander, M. W., Vrana, J. D., Voje, W. E., Carothers, J. M. & Klavins, E. Digital logic circuits in yeast with CRISPR-dCas9 NOR gates. Nat. Commun. 8, 15459 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Liu, Y. et al. Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells. Nat. Commun. 5, 5393 (2014).

    CAS  PubMed  Google Scholar 

  61. 61.

    Chen, P.-Y., Qian, Y. & Del Vecchio, D. In 2018 IEEE Conference on Decision and Control (CDC) 4333–4338 (IEEE, 2018).

  62. 62.

    Chen, Y.-J. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Meth. 10, 659–664 (2013).

    CAS  Google Scholar 

  63. 63.

    Kozuka, K. et al. Development and characterization of a human and mouse intestinal epithelial cell monolayer platform. Stem Cell Rep. 9, 1976–1990 (2017).

    CAS  Google Scholar 

  64. 64.

    Li, H. et al. The outer mucus layer hosts a distinct intestinal microbial niche. Nat. Commun. 6, 1–13 (2015).

    Google Scholar 

  65. 65.

    Fofanova, T. Y. et al. A novel human enteroid-anaerobe co-culture system to study microbial-host interaction under physiological hypoxia. Preprint at bioRxiv (2019).

  66. 66.

    Srinivasan, B. et al. TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20, 107–126 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Shah, P. et al. A microfluidics-based in vitro model of the gastrointestinal human–microbe interface. Nat. Commun. 7, 11535 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Jalili-Firoozinezhad, S. et al. A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat. Biomed. Eng. 3, 520–531 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Roper, J. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat. Biotechnol. 35, 569–576 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank M. Mimee (MIT) and T. Lu (MIT) for providing us with the NBU1-based Bacteroides shuttle vector pNBU1-Erm. We thank M. Fischbach (Stanford University) for providing us with the NBU2-based Bacteroides shuttle vector pNBU2-tetQ. Primary human colonic epithelial cells were obtained by generous contributions from the laboratory of O. Yilmaz (MIT). We thank K. Schneider (MIT) and C. Wright (MIT) for technical assistance. This work was supported by the National Institute of Health P50 grant (P50-GM098792), Office of Naval Research Multidisciplinary University Research Initiatives Program (N00014-13-1-0074), Defense Agency Research Projects Agency Synergistic Discovery and Design (SD2; FA8750-17-C-0229), National Science Foundation Semiconductor Synthetic Biology for Information Processing and Storage Technologies (SemiSynBio; CCF-1807575) program and National Institutes of Health (NIH R01EB021908).

Author information




M.T. and C.A.V. conceived the project and designed experiments. J.Z., Y.H. and L.G. built the in vitro gut model system. M.T., A.T., J.Z. and Y.H. performed the experiments. S.Z. performed the computational work. M.T., S.Z., J.Z. and C.A.V. wrote the manuscript.

Corresponding author

Correspondence to Christopher A. Voigt.

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C.A.V. and M.T. have filed a provisional patent based on this work. All other authors have no competing interests.

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Supplementary Figures 1–24, Supplementary Tables 1–6, and Supplementary File 1

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Taketani, M., Zhang, J., Zhang, S. et al. Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron. Nat Biotechnol 38, 962–969 (2020).

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