An assay for screening an E. coli green fluorescence protein promoter library

Abstract

Biosensors are valuable tools for understanding metabolic pathways and detecting organic molecules, particularly through the use of metabolite-sensing promoters. An important step in the development of biosensors is to create a reliable assay to screen for promoters that respond to metabolites of interest. Although previous studies have explored assay and biosensor development, replicability and feasibility within our current laboratory has yet to be determined.  In this study, we aimed to perform the preliminary steps in developing an assay for screening of an E. coli promoter library by using L-phenylalanine as a model substrate. We obtained a green fluorescence protein promoter library containing 1,820 different promoter regions of E. coli K12 strain MG1655. We performed a pilot study with a subset of the library representing 96 clones to simplify our early stage experiments. Promoter activity was measured as green fluorescence. Heat stress was used as a control condition to assess promoter responsiveness. Serial dilutions of green fluorescence existing clones were performed to test the dynamic range of the assay. Subsequent screens using phenylalanine were done to find promoters responsive to this metabolite. In our subset of 96 clones, we were able to identify five strains that showed altered GFP expression in response to the addition of phenylalanine. Our work here establishes a system to carry out fluorescence-based assays to screen an E. coli promoter library.