Bacteroides species are prominent members of the human gut microbiota—a densely populated and highly competitive environment. The prevalence and stability of Bacteroides in humans make them ideal candidates to engineer as live therapeutics that can be designed to perform specific genetic programs. Here, we develop a framework for transcriptional programming in Bacteroides to facilitate the design of complex genetic circuits. Engineered transcription factors are combined with cooperative genetic architectures to construct transcriptional logic gates dependent on one or two inputs. All fundamental 2-input logic gates are built with &gt;50-fold dynamic range and deployed in several distinct Bacteroides species to demonstrate the universal applicability of this programming system. We then couple transcriptional programming with CRISPR interference to achieve advanced regulation of endogenous genes. By asymmetrically regulating single guide RNAs in different Bacteroides, we demonstrate dynamic control of community composition in co-culture. This work provides a rigorously characterized toolkit to predictably program gene expression in Bacteroides. The resulting technology will enable the development of designer therapeutic bacteria, and provides a method for large-scale, multiplexed perturbation of endogenous genes.

Transcriptional programming in a Bacteroides consortium

ACS Spring 2022

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