Article

Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38, e92

Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544-1014 USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2010; 38(6):e92. DOI: 10.1093/nar/gkp1193
Source: PubMed

ABSTRACT

We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into
arbitrary positions in the Escherichia coli chromosome. The system uses λ-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in
the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering
or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any
orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability
of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.

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    • "However, we were able to achieve 59%, 35%, and 14% efficiency for inserting 3 kb, 5 kb, and 8 kb sequences, respectively. This increased efficiency was possibly attributed to DSB generated recombination stimulation effect (Fig. 2e) (Kuhlman and Cox, 2010). "
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