Multiplexed Genotyping, Tracking and Engineering of Bacteria

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 engineering of biological functions 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 ~10⁵ fold increase in combinatorial library tracking capabilities. Data will be presented tracking population diversity and mapping growth selection dynamics towards engineering alcohol tolerant organisms.