Published: September 1st, 2018
Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.
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.
A common first approach to study the function of a gene is to perform knock-out mutagenesis and observe the resulting phenotype. This is also termed reverse genetics. The bacterium E. coli has been the workhorse of molecular biology for the last 70 years or so, due to the ease of its culturing and its amenability to genetic manipulation1. Several methods have been developed to produce gene deletions in E. coli, including marker exchange mutagenesis2,3 and, more recently, recombineering using the λ Red or Rac ET systems4,
1. Strains and Plasmids
Generation of a tamA Knock-out of BL21ΔABCF:
The strategy outlined above has previously been used to produce a derivative strain of BL21(DE3), a standard laboratory strain used for protein production, which is optimized for outer membrane protein production and called BL21ΔABCF21. This strain lacks four genes coding for abundant outer membrane proteins and, consequently, is able to produce.......
P1 transduction is a fast, robust, and reliable method for generating gene deletions in E. coli. This is demonstrated here by transducing a tamA deletion mutant from a Keio donor strain to a BL21-derived recipient. The major stages in the transduction process are the production of the transducing lysate, the transduction itself, the excision of the Kan resistance cassette, and the verification of the knock-out by PCR. In total, the process takes approximately 1 week and requires no molecular biology met.......
Keio collection strains were obtained from the National BioResource Project (NIG, Japan): E. coli. We thank Dirk Linke (Department of Biosciences, University of Oslo) for his continuing support. This work was funded by the Research Council of Norway Young Researcher grant 249793 (to Jack C. Leo).....
|E. coli BW25113
|Wild-type strain of Keio collection
|E. coli BL21(DE3)
|E. coli BL21DABCF
|Derived from BL21(DE3)
|E. coli JW4179
|tamA deletion mutant
|Generally transducing bacteriophage
|conditionally replicating plasmid with FLP
|for production of EibD; plasmid available on request
|for production of YadA; plasmid available on request
|anti-collagen type I antibody COL-1
|Bovine collagen type I
|Di-sodium hydrogen phosphate
|DNA molecular size marker
|New England BioLabs
|New England Biolabs
|ECL HRP substrate
|goat anti-mouse IgG-HRP
|goat anti-rabbit IgG-HRP
|Skim milk powder
|tamA forward primer
|tamA reverse primer
|Taq DNA polymerase
|New England Biolabs
|Agarose gel electrophoresis chamber
|SDS-PAG electrophoresis chamber
|Wet transfer unit
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