Controlling Heterogeneity and Increasing Titer from Riboswitch-Regulated Bacillus subtilis Spores for Time-Delayed Protein Expression Applications

Sporulated cells have potential as time-delayed expression chassis of proteins for applications such as "on-demand" biologics production, whole cell biosensors, or oral vaccines. However, the desired attributes of high expression rates and low product variances are difficult to maintain from germinated spores. In this work, we study the effect of an integrating vs theta-replicating plasmid in a wild-type Bacillus subtilis and two PolY mutants. The cells were engineered to produce a fluorescent reporter protein (RFP) under the control of a riboswitch activated by theophylline. This allowed for greater sensitivity to point mutations. The fluorescence and cell-growth curves were fit with a custom kinetic model, and a peak kinetic rate (LKP max ) was extracted for each clonal population ( n = 30 for all cell, vector, and growth combinations). Plasmid-based expression yields higher (8.7×) expression rates because of an increased copy number of the expression cassette (10× over integrated). The variance of LKP max values increased 2.1× after sporulation for the wild-type strain. This increase in variance from sporulation is very similar to what is observed with UV exposure. This effect can be partially mitigated by the use of PolY knockouts observed in suspended cell growths and adherent biofilms.