273438 Multiplex Recombineering-Enabled Genome Editing Tools

Monday, October 29, 2012: 3:15 PM
Crawford East (Westin )
Nanette R. Boyle1, Sean A. Lynch2, Thomas J. Mansell3 and Ryan T. Gill1, (1)Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, CO, (2)Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, (3)Chemical and Biological Engineering , University of Colorado at Boulder, Boulder, CO

Recombineering has allowed researchers to rewire genomes much faster than traditional cloning techniques. With the advent of multiplex recombineering, genome-wide editing has become possible, allowing large scale reprogramming of cellular machinery. Despite these successes in large scale genome-wide reprogramming, the overall efficiency of recombineering still remains low, especially for changes > 6bp. Wang et al. have recently reported much higher efficiencies in recombineering by the use of co-selection, which enriches for cells that are highly recombinogenic1. Therefore, we have designed a strain that has co-selection markers located around the genome and are easily screened/selected for, to enable high efficiency recombineering at any location. We have also developed a method to create large libraries of proteins with trackable, targeted changes in just a few days, further enabling genome editing and optimization. We will describe our efforts to apply these new tools for the rapid construction of protein, pathway, and combinatorial genome libraries.  

References

1              Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat Meth advance online publication, doi:http://www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth.1971.html#supplementary-information (2012).


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