Development of a High-throughput Screening Method to Identify Chloride-sensitive Variants of the Fluorescent Protein mPapaya

Engineering genetically encoded chloride-sensitive fluorescent biosensors to understand the behavior of chloride in biological systems is a promising area of research because these biosensors have the ability to target specific locations and the ability to express within specific cells in transgenic organisms while selectively enabling them to determine the behavior of chloride within a cell, both qualitatively and quantitatively. The GFP-derived fluorescent proteins are used in this engineering process as their unique structure enables them to act as anion recognition domain, mainly through the hydrophobic interactions and the formation of hydrogen bonds while the autocatalytic chromophore contributes to generating an optical output as a transducer. Despite the unique properties, these fluorescent proteins exist with certain limitations and therefore need to be modified through mutagenesis to get the desirable features to enable them to function as biosensors. To accomplish this process, detailed structural analysis of the protein structure including chloride binding coordination sphere and chromophore environment is required as the structural analysis will help to identify the sites for introducing mutations. After identifying the potential sites which would foster the desirable features, site-saturation mutagenesis will be used to generate the library. As the mutagenesis library contains a vast number of platforms of mutated variants, including the variants in which our desired feature (chloride-sensitivity) is enhanced/deprived or intact variants with no mutations, there should be a validated method for library preparation, screening and then identifying the chloride-sensitive variants from the library in high-throughput format. This thesis will explain about the development of a workflow to screen chloride-responsive variants in a high-throughput format while specifying every step, from selecting a vector, designing primers, library preparation, screening, and sequencing, as all these steps have a major impact on the protein engineering. We will use mPapaya, as the testing protein and describe a detailed structural analysis to provide a broad picture on selecting positions for engineering the chloride binding pocket in it. The process described in this thesis involves in the identification of an appropriate mutation site through sequence alignment with avYFP-H148Q and the identified site (H202/ p-position) is mutated using site-saturation mutagenesis where the chloride-sensitive variants can be identified based on confidence interval graphs. Future considerations will be to use this method to identify the mutated variants in other testing fluorescent proteins in our lab.