Abstract
Background 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 E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium.ResultsWe 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 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 E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli.ConclusionsIn 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 E. coli. Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0571-y) contains supplementary material, which is available to authorized users.
Highlights
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
We demonstrate the utility of this method by integrating 15 kb DNA encoding sucrose and lactose catabolism pathways and 50 kb of B. subtilis 168 DNA into the E. coli K12 MG1655 chromosome
Results and discussion fliK of the flagellar region 3b as the chromosomal integration target site The potential integration target sites in the E. coli chromosome vary in their suitability for integration of synthetic DNA [16]
Summary
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 E. coli chromosome would greatly facilitate engineering efforts in this bacterium. Escherichia coli K-12 is a gram-negative model bacterium used as a host for a number of synthetic biology and biotechnology applications [1,2,3]. Novel approaches for introducing synthetic DNA, the high molecular weight DNA, into E. coli would greatly facilitate engineering efforts in this bacterium. Integration of synthetic DNA into the E. coli chromosome mitigates against many problems associated with the maintenance of DNA on plasmids or bacterial artificial chromosomes (BACs) [8,9,10,11]. Recent E. coli chromosomal integration approaches include the bacteriophage integrase- mediated recombination between phage attachment (att) sites dubbed clonetegration [12] and the bacteriophage lambda Red recombinase- mediated recombination employing yeast homing mitochondrial I-SceI endonuclease [13, 14], knock-in/knock-out (KIKO) vectors [15] and plasmid pSB1K3(FRTK) [16]
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