Diverse research into the model organism, Escherichia coli, has added substantial depth to our understanding of genome editing of bacteria. Recombineering using the λ Red system is the most disruptive molecular technology discovered thus far, and improved our ability to introduce targeted single nucleotide variants by ~1E4 fold. This discovery has catalyzed incredible progress and enabled ambitious genome/organism engineering projects such as high throughput metabolic engineering to genome-wide codon reassignment. While efforts in E. coli have since accelerated further, work in other bacterial model organisms has lacked this catalyst and continues to fall behind E. coli. To facilitate development of disruptive technologies for non-standard model organisms, we produced a library of homologs to the λ Red recombinase, λ β (NP_040617.1), to generate a toolbox for recombinase discovery in organisms with minimal tools. We demonstrated the recombinase discovery workflow, called Serial Evolutionary Enrichment for Recombinases (SEER), in E. coli and present a number of alternatives to using λ Red for genome editing. We then moved on to explore λ β-mediated recombination in vitro where we able to show that bet specifically unloads E. coli Ssb from Ssb-coated oligos to facilitate annealing. We hypothesized that ssb represents the minimal host interaction node that a recombinase must achieve to facilitate recombination in vivo, and demonstrated a gain-of-function phenotype when species-matched recombinase/ssb pairs are ported into foreign organisms, potentially opening up poorly understood organisms to recombineering using well understood recombinase/ssb pairs.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/14338 |
| Date | 22 January 2016 |
| Creators | Chung, Michelle Y. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0015 seconds