(134d) Engineering Biosensors for Detection and Bioremediation of Chemical Contaminants in a Resistant Soil Bacterium Using an Efficient Genetic Prototyping Approach

Environmental chemical contaminants, such as toxic heavy metals (cadmium, mercury, arsenic, and lead), are prevalent in numerous terrestrial and aquatic environments causing serious deleterious impacts to human health and ecosystems. Detection and biological remediation of contaminants using resistant, living bacterial cells is a potential strategy that could be utilized for water or soil samples. Here, we sought to establish a framework for this approach in the contaminant-tolerant soil bacterium Pseudomonas putida KT2440. We envisioned that the bacterial cells could be programmed using a genetic circuit consisting of a sensor, signal processor, and actuator of biological remediation pathways. Further, distributing these functions for one or more contaminants among strains within programmable bacterial consortia with intercellular communication could improve robustness and mitigate cellular burden. While there are reported biosensor designs utilizing allosteric transcription factors (TFs) responsive to heavy metals (in either P. putida or E. coli), they generally lack the genetic insulation and standardized characterization required to algorithmically design and integrate them into functional circuits.

Therefore, here we sought to create and optimize a library of sensors for a selection of environmental chemical contaminants. We designed, constructed, and characterized biosensors in both E. coli and heavy metal-resistant Pseudomonas putida for 4 contaminants using TFs (CadC, MerR, ArsR, PbrR) and designed cognate promoters and performed an assessment of the transferability of the sensor designs between these bacteria. For rapid sensor design prototyping, we utilized our previously published approach and placed each sensor’s contaminant-responsive TF under the control of a characterized inducible promoter to assay the effects of TF expression on sensor performance without the need for multiple constructs. Fine control of sensor behavior was performed using a few RBS variants. Promisingly, transferring the mercury sensor to P. putida was successful, achieving 250-fold activation, albeit with slightly greater basal activity. To facilitate partitioning and cell-cell communication in an intercellular circuit, we also studied the transfer of sensors for homoserine lactones (HSLs) that were originally engineered for E. coli to P. putida, along with HSL biosynthesis modules. From this work, we established intercellular communication via the Cin, Rhl, and Lux synthase-regulator pairs. The HSL sensor’s characterization further demonstrated their transferability to P. putida albeit with some notable differences in performance. Looking ahead, the sensors developed in this work will be used for environmental detection and integrated into multicellular genetic circuits in P. putida to actuate expression of heavy metal binding domains for bioremediation.