Abstract
Objectives
Genetic modifications to bacterial chromosomes are important for research; recently we reported a two-plasmid system for single locus modification in Escherichia coli and an improved method for simultaneous multiple-loci modification is needed.
Results
An intermediate bacterial strain was generated with different resistance marker genes flanked by I-SceI recognition sites at multiple target loci. Then a donor plasmid carrying several alleles with desired modifications was transformed into the intermediate strain together with a bifunctional helper plasmid encoding λ-Red recombinase and I-SceI endonuclease. I-SceI would induce double-strand breaks (DSBs) in the chromosome and λ-Red would induce recombination between chromosome DSBs and allele fragments from the donor plasmid, resulting in genomic modifications.
Conclusions
This method has been used to successfully perform three different loci modifications simultaneously.
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Acknowledgments
We thank Dr. Jian Li for thoughtful discussions and reading of the manuscript. We thank Benjamin S. Glick (University of Chicago) for providing SnapGene software for producing plasmid maps. We thank Angel Yeast Co. Ltd for providing yeast extract for experiments.
This work was supported by the National Basic Research Program (973 Program) of China [2014CB745100, 2011CBA00800].
Conflict of interest
The authors have declared that no conflict of interests exist.
Supporting information
Supplementary Fig. S1: PCR analysis for cadA deletion, thrBC deletion and gdhA integration.
Supplementary Fig. S2: Lysine production in shake flask.
Supplementary Table S1: Oligonucleotide primers used in this study.
Supplementary Table S2: Plasmids used in this study.
Supplementary Methods and Protocols.
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Yang, J., Sun, B., Huang, H. et al. Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-SceI cleavage. Biotechnol Lett 37, 2011–2018 (2015). https://doi.org/10.1007/s10529-015-1878-1
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DOI: https://doi.org/10.1007/s10529-015-1878-1