(722b) Rapid Engineering of Bacteria with Multiplexed Editing, Testing and Tracking Approaches

Authors: 
Zeitoun, R. I., University of Colorado Boulder
Gill, R. T., University of Colorado, Boulder

Multiplexed genome design and editing strategies can produce targeted libraries containing billions of combinatorial mutations on laboratory timescales. When combined with high-throughput screens or selections, such strategies enable metabolic engineering at scales orders of magnitude beyond prior approaches. A major challenge has been the inability to efficiently and dynamically map such combinatorial library diversity; thus preventing the adoption of more sophisticated engineering strategies that learn from the recursive mapping of a large number of designs onto targeted performance metrics.  In this talk, I will discuss approaches to engineering and tracking combinatorial designed genetic libraries. Specifically, I will cover the development and application of a high-throughput genotyping approach where distal genomic sites targeted by genome-editing methods are copied and assembled into individual DNA constructs that are compatible with next-generation sequencing strategies, thus enabling a ~104 fold increase in combinatorial library tracking capabilities.  We subsequently use this multiplexed editing and testing approach to engineer organisms towards alcohol and biofuel production.  Additional data will be presented tracking population diversity and mapping growth selection dynamics towards rapidly engineering alcohol tolerant organisms.