Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jul 7;336(1):73-80.
doi: 10.1016/j.gene.2004.02.054.

Engineering large fragment insertions into the chromosome of Escherichia coli

Rui Rong  1 , Malgorzata M Slupska, Ju-Huei Chiang, Jeffrey H Miller
Affiliations

Engineering large fragment insertions into the chromosome of Escherichia coli

Rui Rong et al. Gene. .

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.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

Publication types

MeSH terms

LinkOut - more resources