John Bauer – NIH Trainee 2023-2025
Research Progress: I am a chemical engineering PhD student in the Andrews Lab. During the past two years, I have developed my skills in synthetic plasmid design and construction via PCR amplification and Golden Gate Assembly, cloning in both E. coli and P. putida, and fluorescent biosensor assays utilizing flow cytometry. My research is focused on the design of a diverse synthetic microbial consortium featuring multicellular genetic circuits for the bioremediation of toxic wastewater contaminants like Pb2+, Cd2+, As3+, Hg2+, benzalkonium chloride, C6H14, C8H18, and C10H22. This circuit is divided into three modules connected via synthetic bacterial quorum signaling: sensing, regulatory network, and remediation. In the last year, I have made great progress on my first project aim of contaminant sensor construction.
I successfully constructed and characterized allosteric transcription factor-based biosensors for the majority of the contaminants listed above in E. coli. Each sensor features inducible control of the transcription factor to access a range of protein expression to efficiently tune each sensor’s ON/OFF and dynamic range behaviors from a single plasmid construct. I have additionally expanded my biosensor work into P. putida KT2440. KT2440 would be a valuable addition to the bioremediation consortium as it is metabolically robust with high heavy metal and organic solvent resistances (compared to E. coli), and capable of enzymatically degrading benzalkonium chloride. To test its feasibility, I translated previously designed and characterized quorum sensor constructs from E. coli to KT2440. The translation was a great success with all sensor designs displaying comparable ON/OFF and dynamic range behaviors to their E. coli siblings. These initial results pave the way for the incorporation of contaminant sensing and remediating circuit components in P. putida.