The overall goal of the following experiment is to use genome-wide barcoded microbe collections to understand gene drug and gene environment interactions in a highly parallel manner. This is achieved by screening the microbe collection for five to 20 generations of growth in the presence of a drug using robotics to maintain the cells in exponential growth. As a second step, cells are collected, which allows us to extract genomic DNA from 4, 000 to 6, 000 individuals simultaneously.
Next PCR products comprising the amplified barcodes are quantified using either microarray hybridization or barcode sequencing in order to quantify the barcodes, and thereby determine the abundance of each strain in a complex pool. Ultimately, results are obtained that show, which barcoded mutants are sensitive to treatments and are therefore being depleted from the pool during the course of the experiment. The main advantage of the technique we're gonna see today is that rather than dealing with strains individually as colonies on plates or an individual liquid culture here, we can pull tens or hundreds of thousands of different strains and analyze them as one single pool.
So once we realized the power of this assay, we needed to automate it, and fortunately, I was working at the Stanford Genome Technology sensor and was surrounded with engineers that allowed me to accomplish this, Demonstrating the protocol today will be Andrew Smith, a senior graduate student in my laboratory who will walk through all the protocols and associated procedures, as well as Tanya Durick, who will show us how to do the sequencing aspects of the protocol. This protocol utilizes a library of Canada albicans mutants generated by transpose on mutagenesis, where each mutant carries a barcode tag to combine individual mutants into a single pool. A copy of the library is replicated onto 3 84.
Well plates containing edia grow the colonies at 30 degrees Celsius for two to three days until they reach maximal size. Then working in a microbiology environment with flame and sterile LabWare flood each tray with five to 10 milliliters of media and soak for five minutes. Resus, suspend the colonies with a cell spreader and pour the liquid plus cells into a 50 milliliter conical centrifuge tube.
Add DMSO to 7%Adjust the cells to a final OD 600 concentration of 50 per milliliter with media containing 7%DMSO and then aliquot and 40 microliter volumes in PCR strip tubes, pooled cell aliquots can be stored indefinitely at minus 80 degrees Celsius. To begin this procedure, thaw pooled cell aliquots on ice, immediately dilute the pool gently into media with a drug or condition of choice, inoculating at an OD 600 of 0.0625 in a total volume of 700 microliters per well in a 48 well plate. For the purposes of this video, triplicates of two conditions will be prepared.
Include at least one appropriate solvent control on the plate. Seal the plate with a plastic plate seal, grow the cells in a spectrophotometer or a temperature controlled shaker shaking at 30 degrees Celsius with an experimentally determined shaking regimen. For example, shake for 14 minutes, read wells for one minute, then resume shaking.
Part of the cell suspension can be harvested by the robot and saved on a coal plate on the robotic deck. At user-defined generation time points, typically 5, 10, 15, and 20 generations of growth. Harvest at least two od 600 of cells for each sample and time point.
Transfer the harvested cell samples into safe lock micro centrifuge tubes. These cells can then be used for genomic DNA extraction, which will be shown next to begin this procedure. Purified genomic DNA from cells harvested at various time points with a yeast star kit.
According to the manufacturer's instructions with one modification elute, the DNA with 300 microliters of 0.1 XTE. Next, set up two PCR reactions for each sample. One for the UPT tags and one for the down tags.
Check the resulting PCR products on a gel. A 60 base pair product for both PCRs is expected for amplicons, used for hybridization as shown in lanes one and three. In this example, a 130 base pair product is expected for amplicons for barcode sequencing as shown in lanes five and seven.
At this point, the PCR products can either be used for microarray analysis or direct barcode sequencing depending on which PCR product was generated. To prepare for microarray analysis, prewarm the hybridization oven temperature to 42 degrees Celsius and set up a boiling water bath and an ice bucket containing an ice water slurry. Pre-wet the arrays by slowly filling with 120 microliters of one x hybridization.Buffer.
Incubate the arrays in the hybridization buffer at 42 degrees Celsius and 20 RPM for 10 minutes while the arrays are incubating, prepare lock top 0.5 milliliter tubes with 120 microliters of hybridization. Mix per sample plus one extra as a buffer to each tube. Add 30 microliters of uptake, PCR and 30 microliters of down tag PCR to the hybridization mix for total volume of 150 microliters.
Boil for two minutes and set in ice water for at least two minutes. Briefly spin the tubes prior to use. Remove the pre hybridization buffer from the arrays and add 120 microliters of hybridization PCR mix to prevent evaporation.
Cover the array gaskets with a tough spot, hybridize for 16 hours at 42 degrees Celsius and 20 RPM. Remove the arrays from the hybridization oven and remove the tough spots from the chips. Slowly remove the hybridization mix from the arrays by pipette and fill the arrays.
With 120 microliters of wash a load the arrays into the washer of the primed fluidic station. The arrays will be washed using the gene Flex SV 3 4 50 protocol with the following modifications. One extra step with wash A before staining.
Wash B, temperature of 42 degrees Celsius instead of 40 degrees Celsius and staining at 42 degrees Celsius on the fluidic station instead of at 25 degrees Celsius. Following fluidics operations, run the fluidic station, shut down four 50 protocol. Unload the washer after ensuring no air bubbles are present.
Place the arrays in the scanner and scan at an emission wavelength of 560 nanometers. The 130 base pair PCR product is used for next generation barcode sequencing to accomplish this first separate 12 pool DNA samples on a 12%page gel. Next, stain the gel with cyber green and cut out the gel band of interest.
The DNA extracted from the gel is then quantified by realtime PCR generate clusters on a single read flow cell using the CBOT and single read cluster generation kit. Load the flow cell into the sequencer and observe the first base coming off. The sequencer shown here is representative array Data collected at certain points of the protocol.
The pool of tagged tagged mutants was grown for 20 generations in the presence of the antifungal agent clotrimazole and in DMSO as a control log two ratio was calculated and plotted for each gene. All those strains that are significantly sensitive to clotrimazole treatment with a log two ratio of two are highlighted in red. Four strains are shown in this example, NCP one ERG two and two independent alleles of ERG 11.
The known protein target of the antifungal drug clotrimazole strains detected as sensitive in a pooled screen should be verified as to their sensitivity in individual growth assays in the presence of the same concentration of drug. This next graph shows the results of a confirmation experiment here. The four strains detected in the screen had decreased growth relative to the wild type parent strain.
BWP 17 indicated in black. These individual growth curves highlight an important feature of such pooled gene drug screens, which is that the absolute rank of a particular strain does not necessarily reflect its exact level of sensitivity. Furthermore, the results show the value of having multiple alleles for each gene.
In this case, the two ERG 11 disruption mutants have slightly differing sensitivities to the drug. Once mastered, this technique can be done in five days from inoculating the screen through to analyzing the data if performed properly. So while each individual experiment is biologically revealing, the true power comes when you have done a lot of experiments and you've collected a database such that you're now able to predict gene function and drug action.
After watching this video, you should have a good understanding of how to prepare genome-wide barcoded mutant collections for pool screening, and also how to use microray or sequencing to identify genes of interest After its development. This technique allowed researchers in the field of functional genomics to explore the function of each of the 6, 000 different genes in the yeast genome.
