Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Summary

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.

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.

Introduction

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 mutagenesis^2,3 and, more recently, recombineering using the λ Red or Rac ET systems^4,

Protocol

1. Strains and Plasmids

1. Bacterial strains
   1. Use the E. coli strains BW25113⁵, JW4179 (BW25113 tamA::kan)⁷, BL21(DE3)²⁰, and BL21ΔABCF²¹. See Table of Materials for further information.
2. Bacteriophages
   1. Use the phage P1vir for the general transduction. Store the phage as a liquid stock with .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Strains
E. coli BW25113	NIG	ME6092	Wild-type strain of Keio collection
E. coli BL21(DE3)	Merck	69450-3	Expression strain
E. coli BL21DABCF	Addgene	102270	Derived from BL21(DE3)
E. coli JW4179	NIG	JW4179-KC	tamA deletion mutant
P1 vir	NIG	HR16	Generally transducing bacteriophage
Plasmids
pCP20	CGSC	14177	conditionally replicating plasmid with FLP
pASK-IBA2	IBA GmbH	2-1301-000	expression vector
pEibD10	N/A	N/A	for production of EibD; plasmid available on request
pET22b+	Merck	69744-3	expression vector
pIBA2-YadA	N/A	N/A	for production of YadA; plasmid available on request
Chemicals
Acetic acid	ThermoFisher	33209
Agar	BD Bacto	214010
Agarose	Lonza	50004
Ampicillin	Applichem	A0839
Anhydrotetracycline	Abcam	ab145350
anti-collagen type I antibody COL-1	Sigma	C2456
Bovine collagen type I	Sigma	C9791
Calcium chloride	Merck	102382
Chloroform	Merck	102445
Di-sodium hydrogen phosphate	VWR	28029
DNA dye	Thermo	S33102
DNA molecular size marker	New England BioLabs	N3232S
DNase I	Sigma	DN25
dNTP mix	New England Biolabs	N0447
ECL HRP substrate	Advansta	K-12045
EDTA	Applichem	A2937
Glycerol	VWR	24388
goat anti-mouse IgG-HRP	Santa Cruz	sc-2005
goat anti-rabbit IgG-HRP	Agrisera	AS10668
HEPES	VWR	30487
Isopropyl thiogalactoside	VWR	43714
Kanamycin	Applichem	A1493
Lysozyme	Applichem	A4972
Magnesium chloride	VWR	25108
Manganese chloride	Sigma	221279
N-lauroyl sarcosine	Sigma	L9150
Skim milk powder	Sigma	70166
Sodium chloride	VWR	27808
tamA forward primer	Invitrogen	N/A	Sequence 5'-GAAAAAAGGATATTCAGGAGAAAATGTG-3'
tamA reverse primer	Invitrogen	N/A	Sequence 5'-TCATAATTCTGGCCCCAGACC-3'
Taq DNA polymerase	New England Biolabs	M0267
Tri-sodium citrate	Merck	106448
Tryptone	VWR	84610
Tween20	Sigma	P1379
Yeast extract	Merck	103753
Equipment
Agarose gel electrophoresis chamber	Hoefer	SUB13
Bead beater	Thermo	FP120A-115
CCD camera	Kodak	4000R
Electroporation cuvettes	Bio-Rad	165-2089
Electroporation unit	Bio-Rad	1652100
Gel imager	Nippon Genetics	GP-03LED
Incubating shaker	Infors HT	Minitron
Incubator	VWR	390-0482
Microcentrifuge	Eppendorf	5415D
Microwave oven	Samsung	CM1099A
PCR machine	Biometra	Tpersonal
PCR strips	Axygen	PCR-0208-CP-C
pH meter	Hanna Instruments	HI2211-01
PVDF membrane	ThermoFisher	88518
SDS-PAG electrophoresis chamber	ThermoFisher	A25977
Tabletop centrifuge	Beckman Coulter	B06322
Vortex mixer	Scientific Industries	SI-0236
Water bath	GFL	D3006
Wet transfer unit	Hoefer	TE22