An Integrated Approach for Genome-Engineering

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

The overall objective of this proposal is to develop an integrated approach to genome engineering based on the application of synthetic biology and genomics methods pioneered in the laboratories of the investigatory team. The proposed approach brings together advances in DNA synthesis, genomics, and recombineering to enable the quantitative evaluation of billions of rationally designed genetic alterations simultaneously and combinatorially. Specifically, >10,000 unique DNA oligomers are synthesized in parallel, recombined at high-frequency into the Escherichia coli genome, and quantitatively tracked using deep sequencing or molecular barcoding strategies. This process can be performed recursively and automatically in order to rapidly accumulate beneficial modifications into an optimized strain. Here, we propose to apply these advances within the context of engineering a broadly significant model system: the production of biodiesel in E. coli. As part of a larger effort, the platform molecule produced by E. coli will be secreted, extracted, and provided for inorganic catalysis to produce a C12/C14 diesel molecule mixture and a C4 next generation biofuel (i.e.butanol). This approach has the potential to improve economics and carbon-balances compared to alternative biofuels approaches, which should broaden the overall impact of these efforts.