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

BMC Biotechnology

Juhan Kim²  Anthony M Webb¹  Jamie P Kershner²  Stephen Blaskowski¹  Shelley D Copley² 

[1] Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, CO 80309, USA

[2] Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder, CO 80309, USA

关键词: Salmonella enterica;    Escherichia coli;    I-SceI;    Genome editing;   

Others  :  1084559
DOI  :  10.1186/1472-6750-14-84

 received in 2014-05-17, accepted in 2014-08-14,  发布年份 2014

【 摘 要 】

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 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.

Conclusions

The 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.

【 授权许可】

   
2014 Kim et al.; licensee BioMed Central Ltd.

【 图 表 】

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