S24: The Genome Design Shop: Editing, Re-editing and Optimization of Microbial Systems

The ability to comprehensively understand, predict, and modify biological systems at the genome-scale requires the development of new technologies for the iterative search and optimization of mutational-design space on laboratory timescales. A critical barrier here is that even the simplest of desired traits requires the coordinated action of enough biological parts (promoters, regulators, genes, terminators, etc.) that the combinatorial search space rapidly scales to un-addressable sizes.  To confront the difficulties of genome-scale engineering, we bring together an interdisciplinary approach utilizing expertise in recombineering and synthetic biology, systems biology, bioinformatics and biofuels strain engineering to develop a genome-scale technology platform for predictive design, re-design, and optimization of bacterial systems.   Our approach involves two objectives: i) to demonstrate an integrated, genome-scale strategy for “chassis” strain design and construction, and ii) to demonstrate a framework for “genome re-design” built upon multiplex synbio and genome-engineering technologies. The core genome-engineering cycle consists of prototype “chassis” design and construction, Trackable Recrusive Multiplex Recombineering (TRMR)-type library construction and mapping, systems-biology analysis of mapping results and selection of targets for combinatorial libraries, and then construction and evaluation of combinatorial mutant libraries. We have chosen to develop this platform by engineering i) ethylene production and ii) isobutanol production in Escherichia coli. Ethylene and isobutanol are attractive models because both can be directly converted to gasoline (and other advanced liquid biofuels) using well established catalytic routes and both have been produced in E. coli at various levels.