Optimized high-throughput screening of regulatory protein libraries for novel molecular biosensors

In vivo high-throughput screening systems for detecting select metabolites are useful for engineering flux through biosynthesis pathways. Transcriptional regulator proteins serve as gene switches in microorganisms and can be applied as highly specific and sensitive endogenous molecular biosensors. For cases where there is no known regulator that responds to a metabolite of interest, protein engineering may be used to alter inducer specificity of an existing biosensor system. We combine ligand binding pocket saturation mutagenesis with FACS of fluorescent reporter protein expression to generate novel molecular biosensors based on the AraC regulatory protein from E. coli.

Through the course of our experiments we have studied key parameters to improve the design process and streamline high-throughput screening efforts. This talk will highlight findings from these studies.  As examples: Reporter protein toxicity and poorer growth of clones showing more favorable reporter gene expression requires optimization of recovery and culturing of post-sorted clones and preparation for subsequent rounds. We studied the influence of different fluorescent reporter proteins, reporter gene copy number, and various aspects of the AraC regulation platform. Optimizing the time of inducing clones before sorting minimizes occurrence of false positive AraC variants and improves positive screening efficiency. Switching from a dual-plasmid to single-plasmid system amplified on/off expression ratios and improved sorting efficiency. The effect of reducing GFP stability on the stability of fluorescent clones in post-sort populations was also studied. Finally, Next Generation Sequencing (NGS) was used to monitor AraC library sequence evolution, revealing which sort schemes most effectively enrich clones.