title: Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.
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.
  
  
  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 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.
  
  
  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 E. coli.
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK. mj417@cam.ac.uk.
authors.forename: Mario
authors.initials: M
authors.lastname: Juhas
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
authors.forename: James W
authors.initials: JW
authors.lastname: Ajioka
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D013395:Sucrose / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D045522:Recombinases; D013395:Sucrose; D007785:Lactose
keywords: Chromosomal integration; Escherichia coli; High molecular weight DNA; Lambda Red recombineering; Synthetic biology