Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Summary

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Abstract

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Introduction

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease and represents a significant burden to human health. Over 100 million people are infected every year with C. trachomatis¹. Approximately 70% of the infections in women are asymptomatic despite detrimental reproductive health effects, such as pelvic inflammatory disease, ectopic pregnancy, and/or infertility. Disease sequela are directly related to immunopathology initiated by C. trachomatis infection². An efficacious vaccine has yet to be developed; therefore, understanding the function of bacterial virulence f....

Protocol

1. Design and Assembly of pSUmC-4.0 with Homology Arms Specific to the Gene of Interest

1. Identify ~3 kb regions directly upstream and downstream of the gene targeted for deletion to serve as the 5' and 3' homology arms for homologous recombination (Figure 1).
2. Design PCR primers to 1) amplify the 3 kb 5' homology arm from chlamydial genomic DNA and 2) contain a 15–30 bp overhang specific to pSUmC-4.0 when digested at the Sall restriction enzyme site. Ensure that the primer regions overlapping with pSUmC-4.0 have melting temperatures of 55 °C and that hairpin melting temperatures for the entire pr....

Results

The method for markerless gene deletion in C. trachomatis using FLAEM is reliant upon careful cloning and transformation techniques. Successful allelic recombination is an essential first step and requires the identification and insertion of homology arms into the pSUmC-4.0 cloning vector (Figure 1). An essential second step for markerless gene deletion is removal of the fluorescence reporter and antibiotic selection cassette by Cre-lox genome editing, represented in

Discussion

The protocol described here for the generation of markerless gene deletions in C. trachomatis by FLAEM allows targeted deletion of nonessential genes and eliminates cassette-induced polar effects. The protocol relies upon careful design of 5' and 3' homology arms inserted into the pSUmC 4.0 suicide vector, efficient transformation of C. trachomatis, and careful screening of isolated mutant strains. Successful genome engineering via this method results in bacteria that are nonfluorescent and cont.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	KSE Scientific	BMK-A1705	Molecular Biology Grade
Anhydrotetracycline hydrochloride	ACROS Organics	233131000
CaCl2 Buffer			10 mM Tris pH 7.4, 50 mM Calcium Chloride Dihydrate
Calcium Chloride Dihydrate	Sigma	C7902-500G	Suitable for cell culture
Cycloheximide	Sigma	7698-1G
dam-/dcm- Competent E. coli	New England BioLabs	C2925H
DMSO	ATCC	4-X	Sterile filtered cell culture tested
Glutamic acid	Sigma	G8415-100G	L-Glutamic acid
Growth Media #1			RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS).
Growth Media #2			RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS) and 1 µg/mL cycloheximide
Hanks' Balanced Salt Solution (HBSS) (1x)	Gibco	24020-117
Heat Inactivated Fetal Bovine Serum Qualified One Shot (FBS)	Gibco	A38402-02
McCoy Cells	ATCC	CRL-1696
Monarch Plasmid Miniprep Kit	New England BioLabs	T1010S	Small scale DNA purification
NaH2PO4	Sigma	S3139-250G	Sodium phosphate monobasic
Na2HPO4	Sigma	S5136-500G	Sodium phosphate dibasic
NEB 10-beta Electrocompetent E. coli Cells	New England BioLabs	C3020K
NEBuilder HiFi DNA assembly Cloning Kit	New England BioLabs	E5520S	Gibson Assembly Kit
Penicillin G sodium salt	Sigma	P3032-10MU	Bioreagent suitable for cell culture
QIAGEN Plasmid Maxi Kit	QIAGEN	12162	Large scale DNA purification
Q5 Hot Start High-Fidelity DNA Polymerase	New England BioLabs	M0515	Fragment PCR Polymerase
RPMI 1640 Medium (1x)	Gibco	11875-093	Containing 2mM L-glutamine
Sall-HF	New England BioLabs	R3138S
Sbfl-HF	New England BioLabs	R3642S
Selection Media #1			RMPI 10 % FBS, 1 µg/mL cycloheximide, 500 µg/mL spectinomycin, and 50 ng/mL anhydrous tetracycline dissolved in DMSO
Selection Media #2			RMPI 10 % FBS, 1 µg/mL cycloheximide, 500 µg/mL spectinomycin
Selection Media #3			RPMI 10 % FBS, 1 µg/mL cycloheximide, 50 ng/mL aTc, and 0.6 µg/mL penicillin
Sodium Acetate Buffer Solution	Sigma	S7899-100ML	3M
SOC Outgrowth Medium	New England BioLabs	B9020SVIAL
Spectinomycin dihydrochloride pentahydrate, Cell Culture Grade	Alfa Aesar	J61820
Sucrose	Sigma	S1888-1KG	Bioreagent suitable for cell culture
Sucrose-Phosphate-Glutamate Buffer (SPG)			37.5g sucrose, 1.25 g Na2HPO4, 0.18 g NaH2PO4, 0.36 glutamic acid for 500 ml tissue culture grade water
Tris	AMRESCO	0497-5KG	Ultrapure grade
Trypsin-EDTA (1x)	Gibco	25200-056	0.25%
Water	Sigma	W3500-500ML	Sterile-filtered, BioReagent, Suitable for cell culture