Lambda Red recombinase-mediated integration of the high molecular weight DNA into the $\textit{Escherichia coli}$ chromosome

$\textbf{Background}$ $\textit{Escherichia coli}$ K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the $\textit{E. coli}$ chromosome would therefore greatly facilitate engineering efforts in this bacterium. $\textbf{Results}$ We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the $\textit{E. coli}$ chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the $\textit{E. coli}$ chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the $\textit{fliK}$ locus of the flagellar region 3b in the $\textit{E. coli}$ K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of $\textit{Bacillus subtilis}$ 168 DNA into two target sites in the $\textit{E. coli}$ K12 MG1655 chromosome. The chromosomal integrations into the $\textit{fliK}$ locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of $\textit{E. coli}$. $\textbf{Conclusions}$ In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of $\textit{E. coli}$.This work was supported by grants from the UK Engineering and Physical Sciences Research Council, OpenPlant Fund and SynBio Fund.This is the final version of the article. It first appeared from BioMed Central via https://doi.org/10.1186/s12934-016-0571-