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Changing the specificity of Escherichia coli periplasmic binding protein RbsB from ribose towards 1,3-cyclohexanediol and cyclohexanol

Author Diogo Tavares
Director of thesis Jan Roelof van der Meer
Co-director of thesis
Summary of thesis Biosensors have proven to be a powerful and cheap tool for analytics of specific target molecules or conditions (e.g., toxicity). Bioreporter bacteria are genetically engineered to produce a measurable signal (reporter protein) in direct response to a specific chemical or physical agent present in their environment. The design of bioreporter bacteria typically starts with known sensory/regulatory proteins, but it has been proposed that computational prediction of substrate binding interactions in a well-known receptor protein could lead to design of mutant proteins with novel target recognition specificities. Here a mutant library based on the ribose binding protein of Escherichia coli (RbsB) was produced with the goal to find mutations which would permit binding of ribose-similar molecules such as 1,3–cyclohexanediol and cyclohexanol. Rosetta simulation was used to predict critical amino acid residues and potential mutations in the RbsB binding pocket which might permit binding of the two new target molecules. The result was a set of 6 million mutants containing one of 5 possible substitutions at each of 9 amino acid positions. The library was introduced into an E. coli expression strain, which carries a hybrid signaling pathway through which GFP is produced upon binding of the target molecule by the (mutant) RbsB. For better reproducibility, cells were grown as individual microcolonies in alginate beads which were screened by fluorescence activated cell sorting (FACS) for gain-of-function GFP expression in presence of 1,3-cyclohexanediol. Alternate rounds of screening and separation were performed to enrich potential responsive mutants but to eliminate constitutive GFP producers. Since FACS only allows screening of end-points of GFP expression in microcolonies, but not the induction itself, the enriched library is currently being screened by high-throughput microscopy in order to recover potential inducible mutants by 1,3-cyclohexanediol.
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