(129c) A Novel Directed Evolution Approach to Engineering Protein Biosensors with Amino Acid Specificity

Low cost, distributed detection of specific proteins would have imminently useful applications in public health, counter-bioterrorism, agriculture, and environmental monitoring.  However, existing technologies for protein detection are either too expensive and bulky for distributed use (mass spec) or not suitable for continuous monitoring and limited analyte possibilities (immunoassays, i.e., pregnancy test).  A Synthetic Biology-based biosensing solution for specific proteins would require a platform for re-engineering receptors toward different peptide sequences in the extracellular space.  To date, the majority of biosensing parts are (a) intracellular and (b) can not be tuned to detect new analytes.

We have developed a directed evolution approach for tuning sensitivity and specificity of the yeast pheromone receptor (Ste2p) toward peptides.  We have used this approach to evolve receptors that can discriminate one amino acid variants (in a length of 13 amino acids).  The key challenge we overcame was the development of a robust high throughput screen for G-protein coupled receptors (GPCRs) in S. cerevisiae.  The screen relies on GFP reporting of receptor activation and fluorescence-activated cell sorting (FACS).  In optimizing the screen, we demonstrated our two color approach (second color being an internal control) was superior to previous single color screens.  As well, we have developed a Bayesian algorithm that sets optimal sorting thresholds for FACS, and estimates the rounds of enrichments required.  I will discuss the development of the high throughput screen, and the analysis of receptor mutant variants isolated by the screen.  This approach should have many useful applications in Synthetic Biology, including: biosensing, novel cell-to-cell communication (as cells can be easily engineered to produces any number of peptide sequences), and to reveal sequence-activity relationships (SARs) for G-PCRs, a class of drug-target receptors that have been difficult to characterize because of difficulties crystalizing the membrane bound protein.