The overall goal of this procedure is to perform genetic analysis on chlamydia tritus by carrying out chemical mutagenesis and determining genotype phenotype associations. This is accomplished by first generating chlamydia tracom mutant strains using chemical mutagenesis. Next, all chemical generated mutations are identified by whole genome sequencing, followed by reference guided genome assembly.
Then phenotype genotype associations in the mutant strains are identified. Finally, the phenotype genotype associations are confirmed by generating recombinant strains and analyzing them. Ultimately, results can be obtained showing that a mutation in a particular chlamydia gene leads to a phenotype of interest through a combination of classical chemical mutagenesis approaches and next generation Singh.
There are currently no methods available for gene replacement in chlamydia and molecular genetic tools for its study are in their infancy. This method does not require transformation with recombinant DNA or the development of molecular genetic tools yet achieves the same goal. That is an assignment of function to individual gene products.
To prepare cultured cells seat approximately one times 10 of the six vir cells per 25 centimeters squared in three milliliters of DMEM supplemented with 10%FBS incubate at 37 degrees Celsius with 5%carbon dioxide. When the cells reach con fluency about 24 hours later, aspirate the medium and infect the cells with chlamydia at a multiplicity of infection of five. In a total volume of three milliliters of D-M-E-M-F-B-S containing 200 nanograms per milliliter of cycloheximide to begin mutagenesis at 18 hours post-infection under a chemical hood, prepare 20 milligrams per milliliter of ethyl methane sulfonate or EMS in PBS with calcium chloride and magnesium chloride.
EMS is a potent mutagen and carcinogen. Therefore, refer to the text protocol for its disposal. Aspirate the medium from the Vero cells and wash them once with three milliliters of PBS with magnesium chloride and calcium chloride.
Then at three milliliters of EMS solution and incubate for one hour in the fume hood at room temperature. After the incubation, remove the EMS and place it in a container of one molar sodium hydroxide to inactivate the mutagen before washing the cells three times to remove any residual EMS. Then at six milliliters of DMEM 10%FBS with 200 nanograms per milliliter of cyclo heide and 25 micrograms per milliliter of Gentamycin and incubate at 37 degrees Celsius with 5%carbon dioxide for 48 to 72 hours.
Inclusions appear devoid of bacteria about 36 hours after mutagenesis at 72 hours. About 10%of inclusions are filled with bacteria while the rest remain empty to extract elementary bodies or EBS by hypotonic lysis completely aspirate the medium. Add one milliliter of water and rock for 10 minutes to dislodge the cells, lyce the cells by pipetting up and down at least 10 times.
After collecting the lysate in a micro fuge tube, add 0.25 milliliters of five x sucrose, phosphate glutamate buffer, or SPG to a one x final concentration and store at minus 80 degrees Celsius. Set confluent mono layers of ves cells in six well plates by seeding about 0.4 times 10 of the six cells in three milliliters of DMEM 10%FBS per well allow the cells to form a homogenous monolayer over the next 24 hours. Next fall on ice, a stock solution of mutagen chlamydia.
Then prepare six tubes of tenfold serial dilution in a total volume of 200 microliters per dilution in DMEM 10%FBS, and set them on ice. Use three milliliters per well of PBS to wash the Vero cells twice. Then add three milliliters of DMEM to each well for six well plates and 100 microliters of the bacterial dilution in duplicate.
Swirl the plate to ensure even mixture. Spin the infected plates at 2, 700 GS for 30 minutes at 15 degrees Celsius. Then incubate them at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for one to two hours.
In the meantime, prepare 0.54%AROS in DMEM by first combining the following ingredients and mixing. Well then mix in 18.9 milliliters of hot sterile, 1.2%aros in water stirring to prevent clumps. Keep the mixture warm in a 55 degrees Celsius water bath.
Aspirate the bacterial suspension from the dishes and add six milliliters of the warm aro solution. Allow it to solidify completely at room temperature outside the hood. Then in the biosafety hood with the lids removed, dry the plates for an additional 15 minutes when the plates have dried, incubate them at 37 degrees Celsius in a carbon dioxide incubator for seven to 10 days.
Plaques should be visible with the aid of a dissecting microscope one day prior to isolating mutants. See one times 10 to the four zero cells in 100 microliters per well. In a 96 well plate, the cells will be confluent within 24 hours.
Under a dissection microscope, mark the plaques to be picked and then use a sterile one milliliter barrier pipette to collect plugs of plaques. Resus suspend the plaques in 100 microliters of DMEM supplemented with 10%FBS 400 nanograms per milliliter, cyclo, heide, and 50 micrograms per milliliter. Gentamicin to expand the mutant strains, overlay the suspension onto confluent monolayers of Vero cells.
Spin the plates at 2, 700 GS and 15 degrees Celsius for 30 minutes before incubating harvest dbs when greater than 50%of the cells are infected, which can occur as early as 48 hours post infection and as late as 14 days. This quantity of infected cells allows enough viable bacteria to be stored to extract EVs by hypotonic lysis of infected cells. After aspirating the medium at 160 microliters of sterile water and incubate at room temperature for 10 minutes.
Use barrier tips to disrupt the cells by pipetting up and down several times to ensure complete lysis. Finally, transfer 160 microliters of each lysate to micro fuge tubes. Add in 40 microliters of five XSPG and store at minus 80 degrees Celsius until ready to assay and screen mutants for phenotypes of interest.
Exposure to mutagen leads to inclusions that appear devoid of bacteria presumably due to bacterial cell death. Typically, serovar LGVL two will completely lice infected cells within 48 hours post-infection, but treatment with mutagens can extend this cycle to greater than 90 hours, and about 10%of inclusions are expected to recover in our experiments. Infected Vero cells treated with 20 milligrams per milliliter of EMS led to a 99%decrease in the recovery of infectious progeny compared to untreated controls as seen in this figure.
Mutagen treatment also led to the emergence of plaques with altered morphologies, including small plaques. Other morphologies include plaques that appear granular, clumpy, or honeycomb shaped as shown here. The doses of EMS used in these studies can lead to between three to 30 mutations per KCL genome as assessed experimentally by NGS.
Although gradient purified EBS are largely free of host cell material, host DNA is also detected and comprises about 10 to 15%of genomic preps Following this procedure. Screens for phenotypes such as impaired replication in tissue culture cells, loss of biochemical activities or alter bacterial morphologies can be performed in order to answer additional questions like what bacterial factors are required for the formation and maintenance of chlamydia, pathogenic factors.
