Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).
Bibliographical noteFunding Information:
This research was supported by the Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI) [HI13C2163]; Intelligent Synthetic Biology Center of Global Frontier Project [NRF-2012M3A6A8053632]; Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea; Coperative Research Program for Agriculture Science & Technology Development, Next-Generation BioGreen 21 [PJ009034012014] through Rural Development Administration, Republic of Korea. Funding for open access charge was supported by Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea. Jaehwan Jeong is supported by a National Junior Research Fellowship from the National Research Foundation of Korea [NRF-2011-0012414]. This research was supported by the Samsung Advanced Institute of Technology. We thank members of Duhee Bang Lab for their critical comments.
All Science Journal Classification (ASJC) codes