(672b) 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 biosensors1. 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 Fluorescence-Activated Cell Sorting (FACS) of fluorescent reporter protein expression to generate novel molecular biosensors based on the AraC regulatory protein from E. coli, which natively activates gene expression in response to L-arabinose2,3,4.
While our directed evolution approach has furnished new and useful AraC-based biosensors, through the course of experiments we have studied key parameters that could be optimized to improve the design process and streamline high-throughput screening efforts. This talk will highlight the findings from these studies. For example, reporter protein toxicity and poorer growth of clones showing more favorable reporter gene expression required careful optimization of recovery and culturing of post-sorted clones and preparation for the next round. 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 was important to reduce occurrence of false positive AraC variants (leaky or non-specific) and improve the accuracy of the positive screening. Switching from a dual-plasmid to single-plasmid system also amplified on/off expression ratios and improved sorting efficiency. The effect of reducing GFP stability (half-life) through the use of different ssrA degradation tags on the stability of the most fluorescent clones in post-sort populations was also studied. Finally, Next Generation Sequencing (NGS) is being performed to monitor AraC library sequence evolution through different stages of sorting and from different sorting schemes, revealing which sorts most effectively enrich clones.
(1) Gredell, J. A., Frei, C. S., and Cirino, P. C. (2012) Protein and RNA engineering to customize microbial molecular reporting. Biotechnol. J.
(2) Tang, S. Y., Fazelinia, H., and Cirino, P. C. (2008) AraC regulatory protein mutants with altered effector specificity. J. Am. Chem. Soc. 130, 5267–5271.
(3) Tang, S. Y., and Cirino, P. C. (2011) Design and application of a mevalonate-responsive regulatory protein. Angew. Chemie, Int. Ed. 50, 1084–1086.
(4) Tang, S. Y., Qian, S., Akinterinwa, O., Frei, C. S., Gredell, J. A., and Cirino, P. C. (2013) Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J. Am. Chem. Soc. 135, 10099–103.