Engineered Transcription Factors and Logic Gates to Enhance Heavy Metal Biosensor Design in Bacillus Subtilis

Anthropogenic activity including burning of fossil fuels, overuse of pesticides and fertilisers, and the release of contaminated industrial effluent has increased the deposition of toxic heavy metals into the environment, with devastating consequences on human and environmental health. Conventional analytical methods for monitoring heavy metal pollution are hampered by high equipment costs, trained personnel, and inability to quantify bioavailability of contaminants. Bacterial biosensors offer a promising alternative, offering an affordable means of monitoring metals in the environment and providing indications of bioavailable fractions of heavy metals. The well-characterised genetics of the spore-former Bacillus subtilis make it a rational choice for biosensor design, with spores providing the potential for long-term storage. However, design bottle necks such as biological part incompatibility, a lack of heterologous parts, and lack of specificity can severely constrain the design of bacterial biosensors. To address these issues, we report the design of circuits using heterologous chimeric transcription factors based on the MerR family which operate as a Buffer gate, enabling flexible rewiring of circuit specificity through domain swaps and mitigating issues relating to biological part incompatibility. In addition, we also report the exploitation of a natively split-sigma factor system which operates as an AND logic gate, enabling the design of dual-input and ultra-specific heavy metal biosensor circuits. This user-friendly toolbox enables easy biosensor design and will be relevant to other research areas involving synthetic biology such as bioremediation, metabolic engineering and pathogen diagnostics.