Abstract
Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.
Highlights
Lambda Red recombination (‘‘recombineering’’) has emerged as a useful tool in genetics and molecular biology, facilitating the precise generation of insertions, deletions, and point mutations at loci specified by flanking homology regions of as little as 35 bp [1]
Our observations regarding the recombination frequencies of phosphorothioated double-stranded DNA (dsDNA) cassettes [21] led us to investigate the possibility that the ends of cassettes are routinely degraded by endogenous nucleases
An endogenous E. coli nuclease may degrade the 59 PT bonds on one or both strands, thereby allowing Lambda Exo to degrade the remainder of the strand and generate the single-stranded DNA (ssDNA) recombination intermediate
Summary
Lambda Red recombination (‘‘recombineering’’) has emerged as a useful tool in genetics and molecular biology, facilitating the precise generation of insertions, deletions, and point mutations at loci specified by flanking homology regions of as little as 35 bp [1]. Lambda Red, as well as the similar RecET system [2], is capable of modifying Escherichia coli chromosomal [2,3], plasmid [2,4], and BAC [5,6,7,8] targets using either dsDNA cassettes [2,3] or ssDNA oligonucleotides [9,10]. Both strategies have been used toward a broad array of powerful applications. This technique has been used to diversify and rapidly optimize the pathway coding for the biosynthesis of the small molecule lycopene [18], to engineer promoters [19], and to change all E. coli amber (TAG) stop codons into ochre (TAA) stop codons [20]
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