Engineering large fragment insertions into the chromosome of Escherichia coli

Gene
Rui RongJeffrey H Miller

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.

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Citations

Dec 1, 2011·Applied Microbiology and Biotechnology·Daisuke KomaKiyofumi Sakai
Nov 10, 2010·Natural Product Reports·Haoran ZhangBlaine A Pfeifer
Feb 1, 2008·Molecular Pharmaceutics·Haoran ZhangBlaine A Pfeifer
Nov 6, 2021·ACS Synthetic Biology·Zi-Kai WangZheng-Hong Xu

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