Control of bacterial population density with population feedback and molecular sequestration

Genetic engineering technology has become sophisticated enough to allow precise manipulation of bacterial genetic material. Engineering efforts with these technologies have created modified bacteria for various medical, industrial, and environmental purposes, but organisms designed for specific functions require improvements in stability, longevity, or efficiency of function. Most bacteria live in multispecies communities, whose composition may be closely linked to the effect the community has on the environment. Bacterial engineering efforts will benefit from building communities with regulated compositions, which will enable more stable and powerful community functions. We present a design of a synthetic two member bacterial community capable of maintaining its composition at a defined ratio of [cell type 1] : [cell type 2]. We have constructed the genetic motif that will act in each cell in the two member community, containing an AHL-based negative feedback loop that activates ccdB toxin, which caps population density with increasing feedback strength. It also contains one of two ccdB sequestration modules, either the ccdA protein antitoxin, or an RNA device which prevents transcription and translation of ccdB mRNA, that rescues capped population density with induction. We compare absorbance and colony counting methods of estimating bacterial population density, finding that absorbance-based methods overestimate viable population density when ccdB toxin is used to control population density. Prior modeling results show that two cell types containing this genetic circuit motif that reciprocally activate the other's ccdB sequestration device will establish a steady state ratio of cell types. Experimental testing and tuning the full two member community will help us improve our modeling of multi-member bacterial communities, learn more about the strengths and weaknesses of our design for community composition control, and identify general principles of design of compositionally-regulated microbial communities