In vitro Genetic Analysis for Metabolic Engineering of Cell-Free Protein Synthesis

Stanford University, Stanford CA

Investigators

Abstract

The long term goal of this project is to develop methods for efficient and sensitive metabolic surveys of genomes. This project will attempt to use the availability of genomic sequences, massively parallelmolecular biology techniques, and efficient cell-free protein synthesis to obtain functional expression of all the soluble enzymes in a genome, one protein per reaction chamber. With this approach, nearly every enzyme could be tested rapidly for its effect on a targeted metabolic process. This capability would be demonstrated by improving the performance of cell-free protein synthesis. This project will attempt to show that cell-free conditions can be developed to mimic the E. coli cytoplasm and to provide a variety of prosthetic groups so that complex, multimeric proteins will fold properly to become bioactive. This project would also attempt to show that single bioactive proteins can have a profound effect on cell-free protein synthesis and that these effects would be predicted based on the known, intracellular function of the protein. Ten proteins have been chosen to represent various challenges in protein expression and activation. If this project is successful, directed genetic changes could be made with a much higher probability of the desired outcome.