title: Engineering large fragment insertions into the chromosome of Escherichia coli.
abstract: An effective DNA replacement system has been established for engineering large fragment insertions into the chromosome of Escherichia coli. The DNA replacement plasmid, pHybrid I, was first constructed based on the bacterial artificial chromosome (BAC) vector. Two fragments of the E. coli genome, 5.5 and 6.5 kb in length, were introduced into the vector for homologous recombination. In addition to the chloramphenicol gene, a second gene neo was introduced for double marker screening for recombinant clones. By shot-gun cloning and homologous recombination techniques, using our new recombinant vector (pHybrid I), a 20-kb fragment from Lactococcus lactis genomic DNA has been successfully integrated into the chromosome of the E. coli strain J93-140. Plating tests and PCR amplification indicated that the integration remained stable after many generations in cell culture. This system will be especially useful for the chromosome engineering of large heterologous fragment insertions, which is necessary for pathway engineering.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, and The Molecular Biology Institute, University of California, 405 Hilgard Ave., Los Angeles, CA 90095, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Rong
authors.forename: Malgorzata M
authors.initials: MM
authors.lastname: Slupska
authors.forename: Ju-Huei
authors.initials: JH
authors.lastname: Chiang
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Miller
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D004269:DNA, Bacterial