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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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).
The Cello software and codes are freely available (https://github.com/CIDARLAB/cello).
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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).
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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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). https://doi.org/10.1038/s41587-020-0468-5
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