Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.

BMC Microbiology
Gregory J McKenzie, Nancy L Craig

Abstract

Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage lambda red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step. We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion. This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacte...Continue Reading

References

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Citations

Nov 30, 2011·Nucleic Acids Research·Marion H Sibley, Elisabeth A Raleigh
May 19, 2012·Science·Matt Eames, Tanja Kortemme
Jun 19, 2012·Applied and Environmental Microbiology·Sébastien CrépinCharles M Dozois
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Jul 26, 2012·PloS One·Felix BertelsChristian Kost
Mar 22, 2013·PloS One·Sonja BlascheManfred Kögl
Feb 12, 2014·Proceedings of the National Academy of Sciences of the United States of America·Diana BlankOlin K Silander
Apr 1, 2009·Microbiology·Eric D LoVulloMartin S Pavelka
Nov 28, 2012·Biochemical Society Transactions·Katrin Richter, Johannes Gescher
Apr 9, 2015·Molecular Biology and Evolution·Linda De VooghtJan Van Den Abbeele
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Dec 24, 2017·Molecular Systems Biology·Anisha M PerezYousif Shamoo
Jun 17, 2014·Alcoholism, Clinical and Experimental Research·Ashish K SinghGattupalli Naresh Kumar

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Datasets Mentioned

BETA
DQ460223

Methods Mentioned

BETA
PCR

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