Abstract
BackgroundRecently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes.ResultsWe corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands.ConclusionsThe method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations.
Highlights
Introduction of mutations in essential genesIntroduction of mutations in essential genes is difficult because the typical strategy for initial integration of a mutation cassette into the genome disrupts the target gene
We found that pKDTS effectively promoted recombination of the mutation cassette into the genome, as judged by growth of colonies on medium containing the antibiotic for which a resistance gene was carried on the mutation cassette
In the experiment in which we introduced a sequence encoding a C-terminal 3 × FLAG tag on RpoD in E. coli K-12 MG1655 using pSLTS as a helper plasmid, 50% of the colonies obtained on LBAaTc agar plates were wild type when colony purification was not performed after integration of the mutation cassette
Summary
Introduction of mutations in essential genesIntroduction of mutations in essential genes is difficult because the typical strategy for initial integration of a mutation cassette into the genome disrupts the target gene. Developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation This elegantly designed method did not work well in our hands for most genes. The gene gorging method relies upon screening to identify colonies in which a desired genetic change has been introduced by recombination of a mutation cassette into the genome It leaves no scar, but can be inefficient without the use of a special recombination strain [8] or a specially designed synthetic DNA with chemically modified bases [9]. These techniques have been used extensively and modified to enable deletions, insertions, and epitope tagging [5,10,11,12,13,14,15,16,17,18]
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