A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica.

ABSTRACT:

Background

Recently 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.

Results

We 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

SUBMITTER: Kim J 

PROVIDER: S-EPMC4236582 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature