(26b) Development of Transcriptional Logic Gates in Bacillus Subtilis for Predictive Design of Genetic Circuits in Programmable Bacterial Consortia

Genetic circuits enable programming dynamic cellular responses to multiple signals in living cells. Libraries of transcriptional logic gates and algorithms for predictive design of genetic circuits have been developed to temporally control biological functions in Escherichia coli and some other microbes, yet only in a small subset of bacteria. Here, we sought to enable the construction of programmable genetic circuits in an industrially-relevant and Gram-positive Firmicute, Bacillus subtilis. In this study, we developed and learned design rules for creating transcriptional NOT gates in B. subtilis, which can be used to build synthetic genetic circuits. Starting with a set of 6 orthogonal TetR-family repressors, we varied different genetic elements of the NOT gates, including the cognate output promoter sequence and the repressor protein expression. Output promoter sequences transferred from E. coli generally had little activity in B. subtilis. To study the effect of the location of the operator placement on the NOT gate functionality, we designed synthetic promoters for B. subtilis by placing each operator sequence within a core promoter either in the transcriptional start region (TSR), upstream of the −35 region, or overlapping the core promoter elements (−35, −10, and/or −16 regions). We also varied the number of operator sequences in the TSR and observed significantly greater repression for a subset of the repressors. To determine the effect of the repressor’s expression level on each NOT gate response, we expressed the repressor with different inducible promoters and synthetic ribosome binding sites and assayed the library of synthetic NOT gates. In this work to date, we have developed a library of 13 transcriptional NOT gates (dynamic range up to 150-fold and at least 20-fold for each) containing 5 different repressors, and ongoing efforts are working to expand this library. In previous work from our group, we developed synthetic homoserine lactone sensors for B. subtilis intercellular communication. Looking ahead, this genetic NOT gate toolbox can facilitate the design of synthetic genetic circuits for B. subtilis and programming dynamic behaviors in bacterial consortia using this platform.