This protocol describes a simple and effective suppressor screen to identify suppressor mutations in mutants that have a growth defect in fission yeast. Traditional mutagenesis methods use toxic chemicals or UV that can generate multiple mutations in a cell. Conversely, this protocol can enrich a single suppressor mutant in individual cells without using mutation inducing methods.

Begin this procedure with strain construction and preparation as described in the text protocol. Add 200 microliters of liquid media to each well of a 96-well polystyrene microplate. With a sterile applicator, take a small amount of each of the prepared colonies and suspend each colony in individual wells that contain the liquid media.

Include a blank well for every row on the plate containing 200 microliters of the same media. Now set up the protocol on the plate reader detection software connected to an automated microplate reader. Set the temperature at 30 degrees Celsius and set a kinetic program for 24 hours with reading frequency of two minutes.

This results in 721 total reads over a 24-hour period per well. Set the shaking to continuous fast orbital shaking. Set the optical reads to measure light scatter at a wavelength of 600 nanometers to measure optical density and set the light to read from below the plate.

After 24 hours, record the final blank optical density readings and use the formula in the text protocol to determine the volume needed to dilute each of the samples down to an optical density of 0.1. To batch process the dilution volume to be used from each experimental well, export the data from the plate reader software and use a spreadsheet software to insert the same formula as a function. Every 24 hours, use the same media as day zero to dilute each of the samples to an optical density of 0.1 using the formula.

Make sure to use the formula to dilute all wells down to an optical density of 0.1. This includes the wells that may have started showing some recovery in their growth defect. Save all growth curves generated daily.

Note any individual colony that shows an increased growth rate judged by a final optical density that is significantly higher than the rest of the cohort with the same genetic background or by a growth curve that is similar to that of wild type colonies. This assay usually takes about seven to 14 days. Perform all steps under sterile conditions.

From the last day of the plate reader assay, some liquid cultures have a noticeably recovered growth rate presumably by gaining a suppressor mutation that can alleviate the phenotype of the parental mutation. To store the phenotypically recovered cultures, transfer and mix 250 microliters of liquid culture to a cryotube containing 250 microliters of 50%glycerol. Flash freeze the cells in liquid nitrogen and save the strains in minus 80 degrees Celsius indefinitely.

To confirm that the suppressor mutation is a genetically heritable element, use standard genetic crossing methods to cross your favorite mutant P cells with your favorite mutant S cells. When two haploid cells with a complimentary mating type are mixed and subjected to nitrogen starvation, they can generate a zygote that sporulates to form a tetrad of four spores. The parental genetic materials will segregate during meiosis following the rules of Mendelian genetics.

After sporulation, individual spores from the same tetrads can be dissected and form four individual colonies as observed vertically on a plate. Evenly place the spores one by one on the plate using a microneedle. After dissection, leave the cells to grow in a 30 degree Celsius incubator for a few days until colonies appear.

If the suppressor mutation is a genetically heritable element, this cross should yield tetrads in which two colonies have the sickness phenotype of the parental strain and two colonies have the recovery growth rate of the suppressor strain. Pick three colonies with the suppressor phenotype and three colonies with the parental phenotype from the same genetic cross and proceed with the genomic DNA extraction and sequencing steps as detailed in the text protocol. Then perform bioinformatics analysis for the identification of the suppressor mutations as detailed in the text.

Growth curves of individual colonies were recorded at the initial time point and for six days with continuous monitoring using the plate reader. As expected, wild type colonies show no noticeable changes in their growth curves throughout the experiment. Notably, four colonies with the elf1 deletion background and one fal1 deletion colony show a dramatic shift in growth from slow growing to some varying levels of growth similar to that of wild type colonies.

Dramatically, all clr6 mutants show a consistent phenotypic recovery growing at a faster rate by the end of the assay. Two of the identified non-synonymous changes, the cue2 mutation and the rpl2702 mutation were reconstructed in the lab using standard protocols for site directed mutagenesis. Cue2 elf1 double mutants and rpl2702 elf1 double mutants were crossed with the complimentary elf1 deletion mutant strain.

Genetic crossing showed that the identified suppressor mutations are successful in suppressing the slow growing phenotype of elf1 deletion mutant and are heritable. During the daily dilution of the 96-well plate, it's important to dilute all wells to the same concentration in order to avoid artificial growth recovery. This method allows the isolation of suppressor mutations in a high throughput manner speeding up the discovery of hundreds of genes that have not been well characterized in fission yeast and other microorganisms.

This method can be used to isolate suppressors for all microorganisms that have mutation causing growth defect and that can be grown to a large population in liquid culture. Identifying suppressor mutations in fission yeast could have potential implications in finding disease-causing genes in overlapping pathways especially when it comes to highly conserved pathways from yeast to humans.

요약

더 많은 비디오 탐색

Fission Yeast

Schizosaccharomyces Pombe