Establishing a Dual Channel System for Ratiometric Characterisation in Bacillus Subtilis Across Conditions and Gene Contexts

Bacillus subtilis is an industrially important GRAS organism and is a useful chassis for a number of applications within synthetic biology. To facilitate the use of Bacillus, we have developed improved methods for characterisation of gene expression systems.

Dual channel fluorescent reporter systems use two spectrally distinct fluorescent proteins to improve the characterisation of a gene expression system for downstream engineering. By normalising the output of the expression system under investigation to that of a consistent reference, we can distinguish between the effects of intrinsic factors (e.g. sigma-factor affinity) and extrinsic factors (e.g. global metabolic capacity) on the test system.

Here we establish and demonstrate the utility of a dual reporter in Bacillus subtilis. Developing a cloning strategy based on Golden Gate cloning to quickly insert a desired query sequence upstream of the relevant fluorescent reporter allowed us to quickly generate test strains for ratiometric analysis. We screened a range of promoters and RBSs with varying activities, and compared the measured activity across different growth conditions and integration loci. While previous ratiometric work uses bulk assays via a plate reader, we used flow cytometry to gather single-cell data which gives a more complete picture of the activity of individual cells within a population.

This work demonstrated that measurements made by ratiometric characterisation in Bacillus subtilis were more reliable and practical for engineering than single channel measurements. The technique allows the expression system, locus and growth condition to be characterised largely independently reducing the combinatorial cost, as well as automatically correcting for the effect of the reporter system on the host metabolism. Single-cell measurements conducted by flow cytometry also showed that dual channel characterisation allowed us to account for population heterogeneity.

This study demonstrates that dual channel characterisation provides significant advantages in accurate part characterisation, even in systems where gene copy number is more tightly controlled than the plasmid systems where dual channel reporters first emerged.