| The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.;In Chapter 1, I discuss the capabilities of Lambda Red recombination, and introduce its notable past uses, particularly in genome engineering. I also summarize the current mechanistic understanding of Lambda Red, describe several past improvements of the recombination process, and discuss our motivation for improving Lambda Red recombination further.;In Chapter 2, I advance and support a novel mechanism for the Lambda Red recombination of dsDNA, in which Lambda Exonuclease entirely degrades one strand, leaving the other strand intact as ssDNA. This single-stranded intermediate then anneals at the lagging strand of the replication fork and is incorporated into the newly synthesized strand. In Chapter 3, I use this mechanistic insight to investigate new methods for improving dsDNA recombination frequency, finding that recombination frequency can significantly be enhanced by using phosphorothioate bonds to protect the 5' end of the lagging-targeting strand, and by removing the endogenous nuclease ExoVII.;Chapters 4 and 5 detail my efforts to improve the multiplex recombination of short oligonucleotides. In collaboration with Marc Lajoie and Christopher Gregg, I find that removing a set of five exonucleases results in significantly higher multiplex oligonucleotide recombination frequencies, as well as the improved inheritance of mutations carried on the 3' ends of oligonucleotides. Additionally, multiplex recombination frequencies were further improved by modifying DnaG primase so as to increase the amount of accessible ssDNA at the lagging strand of the replication fork.;Finally, in Chapter 6, I suggest additional ways in which Lambda Red recombination may be improved in the future, and discuss a recent project that illustrates how the developments described in this thesis have enabled exciting new applications. |
