Producing gene deletions in Escherichia coli by P1 transduction with excisable antibiotic resistance cassettes

Saragliadis, A., Trunk, T. and Leo, J.C. ORCID: 0000-0002-7066-7527, 2018. Producing gene deletions in Escherichia coli by P1 transduction with excisable antibiotic resistance cassettes. Journal of Visualized Experiments (JoVE) (139): e58267. ISSN 1940-087X

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Abstract

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Item Type: Journal article
Publication Title: Journal of Visualized Experiments (JoVE)
Creators: Saragliadis, A., Trunk, T. and Leo, J.C.
Publisher: JoVE
Date: 2018
Number: 139
ISSN: 1940-087X
Identifiers:
NumberType
10.3791/58267DOI
Divisions: Schools > School of Science and Technology
Depositing User: Jill Tomkinson
Date Added: 19 Jun 2019 09:42
Last Modified: 09 Jan 2020 03:00
URI: http://irep.ntu.ac.uk/id/eprint/36858

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