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.
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"Although several modified E. coli strains (EcNR2, DY330, and EcHW24) are available for genome engineering purposes –, these strains possess several disadvantages including disordered cell growth owing to the cytotoxic genes of defective prophage (like kill) ,  and a permanently inactivated MMR system that results in significant accumulation of undesired background mutations , , , . In addition, the defective prophage is not easily portable to different E. coli platforms, considerably hindering the full exploitation of MAGE functionality for extensive genome editing. "
[Show abstract][Hide abstract] ABSTRACT: Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.
PLoS ONE 04/2014; 9(4):e94266. DOI:10.1371/journal.pone.0094266 · 3.23 Impact Factor
"Several modifications have been made to λ-Red recombineering to improve recombination frequency that can otherwise be a major limitation in certain applications. These include using transformation amplimers with large flanking regions , the removal of endogenous nucleases from the host strain , and the use of three-way overlapping PCR methods that negate the need for multiple intermediary cloning steps , . Complementation of a specific mutation, a common procedure used to prove the function of a gene and fulfill Koch's postulates, is normally achieved in trans by introduction of the relevant gene into the cell on a plasmid. "
[Show abstract][Hide abstract] ABSTRACT: Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.
PLoS ONE 04/2014; 9(4):e94786. DOI:10.1371/journal.pone.0094786 · 3.23 Impact Factor
"This mutation, dnaGQ576A, resulted in 63% greater allelic replacement per clone than the wild type. A follow-up study removed five endogenous exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo); these mutations alone increased recombineering efficiency 46% and when combined with the dnaGQ576A mutation resulted in 111% more clones per cycle than the wild type; however, these mutations came at the expense of cellular growth rate  . "
[Show abstract][Hide abstract] ABSTRACT: Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, which can be divided into three main areas: (i) optimizing the oligo used for recombineering to enhance replication fork annealing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increased rate of annealing; and (iii) multiplexing recombineering targets and automation. These efforts have increased the efficiency of recombineering several hundred-fold. One area that has received far less attention is the problem of multiple chromosomes, which effectively decrease efficiency on a chromosomal basis, resulting in more sectored colonies, which require longer outgrowth to obtain clonal populations. Herein, we describe the problem of multiple chromosomes, discuss calculations predicting how many generations are needed to obtain a pure colony, and how changes in experimental procedure or genetic background can minimize the effect of multiple chromosomes.