(174am) Engineering Whole Cell-Based Microbial Biosensors for Detection and Bioremediation of Chemical Contaminants Using an Efficient Genetic Prototyping Approach

Human activities have led to a marked and hazardous prevalence of chemical contaminants in numerous terrestrial and aquatic environments causing serious deleterious impacts to human health and ecosystems. These chemical contaminants include heavy metals, such as cadmium and mercury, and organic chemicals including alkanes and quaternary ammonium compounds. Current methods for assaying these chemicals are effective but laborious and require expensive instruments staffed by trained professionals. Detection and bioremediation of toxic water contaminants via living bacterial cells containing programmed genetic circuits is an attractive potential alternative. Genetic circuitry consists of sensor, signal processing, and actuation modules. Distributing these functions among cells within programmable bacterial consortia could offer additional advantages and mitigate cellular burden on the host cell due to heterologous expression. There is a wealth of previously reported whole-cell biosensors employing allosteric transcription factors (TFs), including those for chemical contaminants. However, nearly all these sensors lack sufficient insulation in their genetic designs and standardized performance characterization, which is required to integrate these sensors as modules in functional circuits using design algorithms. Therefore, here we sought to create and optimize a library of sensors for a selection of environmental chemical contaminants. We have designed, constructed, and characterized biosensors in Escherichia coli for 6 contaminants using the TFs CadC, MerR, AlkS, and QacR. Our constructs feature insulating terminators and self-cleaving ribozymes that allow for the characterization of their output promoters in relative promoter units, facilitating signal matching during larger circuit design. For rapid sensor design prototyping, each sensor’s contaminant-responsive TF was placed under the control of an inducible promoter to assay a range of genetic expression during sensor tuning without the need for additional constructs. We are also exploring the transferability of these biosensors to other Gammaproteobacteria that offer high chemical tolerance using broad-host replicative plasmids, along with sensors for programmable communication and intercellular signaling. Looking ahead, the sensors developed in this work could be used for environmental detection and integrated into multicellular genetic circuits for bioremediation.