Bacillus subtilis is a valuable industrial production platform for proteins, a bacterial model for cellular differentiation and its endospores have been proposed as a vehicle for vaccine delivery. As such B. subtilis is a major synthetic biology chassis but, unlike Escherichia coli, lacks a standardized toolbox for genetic manipulation. EcoFlex is a versatile modular DNA assembly toolkit for E. coli synthetic biology based on Golden Gate cloning. Here we introduce BacilloFlex, an extension of the EcoFlex assembly standard to B. subtilis. Transcription units flanked by sequences homologous to loci in the B. subtilis genome were rapidly assembled by the EcoFlex standard and subsequently chromosomally integrated. At present, BacilloFlex includes a range of multi-functional B. subtilis specific parts with applications including metabolic engineering, biosensors and spore surface display. We hope this work will form the foundation of a widely adopted cloning standard for B. subtilis facilitating collaboration and the sharing of parts.

Arsenic contaminated ground water is a serious public health issue, and recent estimates place 150 million people worldwide at risk. Current chemical field test kits do not reliably detect arsenic at the lower end of the relevant range, and may generate toxic intermediates and waste. Whole-cell biosensors potentially provide an inexpensive, robust and analyte-specific solution to this problem. The second generation of a Bacillus subtilis-based arsenic biosensor, designated Bacillosensor-II, was constructed using the native chromosomal ars promoter, arsR and the reporter gene xylE encoding catechol-2,3-dioxygenase. Within four hours, Bacillosensor-II can detect arsenic in the form of arsenate (AsO4 3-) at levels more than one order of magnitude below the recommended safe limit for drinking water suggested by the World Health Organisation (10 µg/L). Detection is reported by the enzymatic conversion of the inexpensive substrate catechol to 2-hydroxy-cis,cis-muconic semialdehyde, a bright yellow product visible by eye. We hope that this work will aid in developing a simple inexpensive field test kit for screening of drinking water for arsenic contamination.

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.