Carbohydrate-recognizing regulators from Ruminiclostridium thermocellum work orthogonally in Bacillus subtilis

Orthogonal regulation is important for the field of synthetic biology in order to reduce cross-talk between endogenous and recombinant regulators in engineered microbes. My goal for this project was to determine the feasibility of using a carbohydrate sensing regulatory system from the thermophilic bacterium Ruminiclostridium thermocellum in Bacillus subtilis. Both Firmicutes evolved from a common ancestor; however, this regulatory system evolved in R. thermocellum and not in B. subtilis, making it a candidate for orthogonal regulation. If successful, this system will be used to construct a biosensor microorganism, using B. subtilis, that will respond to plant-derived carbohydrates such as cellulose, xylan, and pectin, by producing green fluorescent protein (gfp). R. thermocellum anti-sigma and sigma (σ) factors will be cloned into a replicating B. subtilis plasmid using isothermal assembly, while the cognate promoter will be cloned in front of gfp on a non-replicating plasmid using Golden Gate assembly. We modified an existing non-replicating plasmid for use in Golden Gate cloning, including replacing the strong lactose-inducible promoter with a lacZ cassette flanked by BsaI sites and using site directed mutagenesis to remove a BsaI recognition site from the β-lactamase gene. Both plasmids were transformed into supercompetent B. subtilis SCK6. Using a spectrofluorimeter, we quantified levels of non-target expression from the R. thermocellum promoter in B. subtilis. Constructed plasmids and engineered B. subtilis strains will ultimately be used to construct biosensors for use in directed evolution of carbohydrate recognizing proteins.