Floxed cassette allelic exchange mutagenesis is an important method because gene deletion is a valuable tool that can be utilized to understand the function of gene products. The main advantage of this technique is that it allows targeted gene deletion without inflicting polar effects on downstream genes. Identify approximately three kilobase regions directly upstream and downstream of the gene targeted for deletion to serve as the five prime and three prime homology arms for homologous recombination and assign primers as indicated in the text protocol.
Amplify the three kilobase five prime and three prime homology arms from freshly extracted chlamydial genomic DNA by PCR in one or two reactions to yield enough fragment for later DNA assembly. After purifying and concentrating the PCR fragments as described in the text protocol, digest 500 nanograms of pSUmC-4.0 vector in a 20 microliter reaction with one unit of cell one and one unit of SBF one according to the manufacturer's protocol. Combine the digested and purified pSUmC-4.0 vector and both the five prime and three prime homology arm PCR products together at a ratio of 0.01 picomoles vector to 0.09 picomoles of each homology arm.
Assemble the fragments in a DNA assembly reaction according to the manufacturer's protocol. Transform 50 microliters of electrocompetent E.coli with one microliter of the DNA assembly reaction. Plate the transformed E.coli onto LB agar plates containing 100 micrograms per milliliter spectinomycin.
Incubate the plates at 37 degrees Celsius overnight. The next day, screen colonies for green and red fluorescence using an epifluorescence inverted microscope. A total of five to 30 colonies per agar plate is expected.
Following an overnight liquid culture incubation of the selected colonies, confirm the insertion of each homology arm into the vector using the PCR screening primers to amplify each insertion. Seed McCoy cells which are mouse fibroblasts typically used for cultivating Chlamydia into two six-well plates as described in the text protocol. In a 1.5 milliliter tube, add C.trachomatis wild-type serovar L2 elementary bodies, or EBs, at a volume that is sufficient to infect 12 wells of a six-well plate at an MOI of two.
Pellet the EBs at greater than 20, 000 times g for 30 minutes and discard the supernatant. Initiate chlamydial transformation by gently resuspending the EBs in 600 microliters of calcium chloride buffer. Then, add 12 micrograms of pSUmC-4.0 plus homology arms to the tube and gently mix the solution by flicking.
Incubate the tube at room temperature for 30 minutes flicking to mix every 10 minutes. Add the transformation solution to 24 milliliters of HBSS and mix by gently pipetting. Transfer two milliliters of the inoculum to each well of confluent McCoy cells in the six-well plates.
Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Remove the inoculum and replace with two milliliters per well RPMI growth media number two. Incubate the plates at 37 degrees Celsius with 5%carbon dioxide.
After a minimum of seven hours but no more than 12 hours, replace the media with two milliliters per well of selection media number one. Continue the incubation at 37 degrees Celsius for 48 hours from the time of infection. Harvest and passage the bacteria onto a fresh monolayer of McCoy cells by using a cell scraper to gently scrape the McCoy monolayer to lift the cells into the media.
Transfer the material from each well into a two milliliter tube. Pellet the cell material at greater than 20, 000 times g in a microcentrifuge for 30 minutes at four degrees Celsius. After discarding the supernatant, resuspend the cell pellet in one milliliter of HBSS by gently pipetting.
Pellet the cell debris at 200 times g for five minutes at four degrees Celsius. Transfer the supernatant into a fresh well of confluent McCoy cells. Add an additional one milliliter of HBSS to each well for a total volume of two milliliters per well.
Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Replace the inoculum with selection media number one immediately following infection. Continue passaging the bacteria onto a fresh monolayer of McCoy cells every 48 hours until red and green inclusions are detected using an epifluorescence inverted microscope 24 hours post-infection.
Once red and green inclusions are detected, continue passaging the monolayer as before using selection media number two. Continue passaging the monolayer until green-only inclusions are detected 24 hours post-infection which indicates the loss of the pSUmC-4.0 vector and incorporation of the loxP selection cassette into the genome. Harvest and freeze the green-only inclusions in SPG.
Enrich green-only inclusions to an MOI of between 0.5 and 1.0 and harvest the monolayers into SPG as described in the text. Obtain aliquots of 50 microliters in 1.5 milliliter tubes and freeze at minus 80 degrees Celsius. Seed a 384-well tissue culture plate with McCoy cells as described in the text protocol.
Dilute an aliquot of green-only bacteria in HBSS to achieve approximately 50 EBs per 20 milliliters. Transfer the diluted inoculum to a reservoir tray. Remove the media from the 384-well plate of confluent McCoy cells by firmly flicking the entire plate upside down into a waste container.
Using a multichannel pipette, add 50 microliters of C.trachomatis inoculum to each well. Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Remove the inoculum by flicking and replace with 50 microliters per well of growth media number two.
Incubate the plate at 37 degrees Celsius with 5%carbon dioxide. After incubating the plate for five to 12 days, identify individual wells with green fluorescent inclusions using an epifluorescence inverted microscope or a high-content screening platform. Harvest wells with green-only inclusions by scraping the monolayer with a P10 tip.
Transfer the entire contents of the well into a tube containing two milliliters of HBSS. Gently mix and apply the two milliliters of inoculum to a fresh confluent McCoy monolayer in a six-well plate for infection. Enrich, freeze, and titer clonal populations.
Confirm the deletion of targeted genes from clonally isolated C.trachomatis using quantitative PCR. Repeat the transformation process using the clonally isolated C.trachomatis FRAEM mutant, pSU-Cre vector, and selection media number three. Passage the monolayer until red-only inclusions are detected which indicates loss of the selection cassette.
Clonally isolate red-only inclusions by limiting dilution in a 384-well plate as before. Enrich clonal populations by passaging until an MOI of 0.1. Once enriched, replace the selection media with growth media number two to initiate the loss of the pSU-Cre vector.
Clonally isolate non-fluorescent bacteria as before. However, manually scan the plate with a Brightfield microscope to detect inclusions. Enrich and freeze the bacteria.
Screen for the mutant strain using quantitative real-time PCR, Western blotting, and whole genome DNA sequencing as previously described. Representative data is shown in which tmeA is targeted for gene deletion. The C.trachomatis tmeaA deletion strain is generated using FRAEM and the C.trachomatis tmeA-lx strain is generated using FLAEM.
Relative DNA copy numbers of tmeA and downstream tmeB, gfp contained on the pSUmC-4.0 selection cassette, and cre contained on the pSU-Cre vector are all shown. Both mutant strains contain a deletion of the tmeA locus. However, tmeA-lx does not contain the selection cassette as indicated by the absence of gfp DNA.
The tmeA mutant strain has decreased expression of tmeB as shown by mRNA and protein levels. When FLAEM is used to generate the tmeA-lx mutant strain, that expression of tmeB is restored as detected by mRNA and protein levels. Careful construction of the pSUmC vector with homology arms is essential for targeted deletion of genes.
Time should be taken to ensure accurate vector assembly prior to chlamydial transformation. FLAEM has provided researchers a tool to study specific genes and make targeted deletions without having decreased expression of off target genes which may confound studies. C.trachomatis deletion mutants generated by FLAEM can be utilized to study a variety of aspects of chlamydial biology including developmental and pathogenic mechanisms.
