The overall aim of this procedure is to generate strains of yeast carrying multiple deletions marked with a green fluorescent protein reported gene. This is accomplished by first preparing haplo single mutants termed pro monsters, each of which carries A GFP deletion cassette in place of one of the target genes. The second step is to assort the deletions via mating and porion of yeast strains using two sets of diploid and haplo selection markers termed GM toolkit A and GM toolkit alpha, that are also present in the pro monster strains.
Next, highly fluorescent cells are selected from the myotic population using flow cytometry to enrich for strains carrying two deletions. The final step is to confirm the genotypes of the sorted strains. Ultimately repeated rounds of mating sation and flow cytometric enrichment, starting with resulting strains lead to the generation of higher orum multi deletion strains.
The main advantage of this technique over existing methods like the G method for marker reuse and sequential gene deletion, is that multiple deletions can be acquired per cycle, resulting in quicker assembly of multi mutants Following generation of pro monsters as described in the written protocol accompanying this video begins sexual cycling by preparing cultures of the established pro monster GFP deletion strains in a 1.6 milliliter eend orph tube. Prepare deletion, strain of mating type A in 100 microliters of synthetic complete medium lacking histamine referred to as minus his medium. Also prepare deletion strain of mating type alpha in 100 microliters of synthetic complete medium lacking leucine or minus lu medium in a 1.6 milliliter eend orph tube for air exchange.
Make holes in the caps of the tubes using a push pin treated with 70%ethanol. Rotate the tubes horizontally at 30 degrees Celsius overnight. The axis of the rotation needs to be parallel to the longitudinal axis of the tube.
Estimating from optical density mixed roughly 250, 000 a haplo cells and 250, 000 alpha haplo cells of GFP deletion strains in a 1.6 milliliter einor tube. This cell number usually corresponds to seven microliters of each overnight culture vortex. The cells proceed to make these cells by first centrifuging at 800 G for two minutes.
After removing the supinate, make a hole on the lid using a push pin treated with 70%ethanol. To allow for air exchange, add 50 microliters of YPDA medium following a quick vortex centrifuge your gain at 800 G for five minutes. Then place the tube in a damp box created with an empty tip box, a small tip stand, and a wet paper towel.
Incubate the box overnight at 30 degrees Celsius. Transfer 10 microliters of the mating mixture to 500 microliters GNA medium without agar containing antibiotics G 4 1 8 and nat in a five milliliter culture tube with a loosened lid, the starting optical density has 600 nanometers is roughly 0.1. Rotate the culture at 30 degrees Celsius for 24 hours.
This allows for the selection of diploids because only diploids containing both can MX four in GM toolkit A and nat MX four. In GM toolkit Alpha can grow in the presence of G four 18 and Nat transfer 20 microliters of the one day culture to 500 microliters of fresh GN medium containing G four 18 and net. The starting optical density at 600 nanometers is roughly 0.2.
Rotate the tube at 30 degrees Celsius for five hours to bring cells to the log phase. To s, correlate the diploids and isolate spores. Follow the procedure outlined in the written protocol.
Proceed to split the suspension of SPO related cells into two 300 microliter parts and put each into a separate 1.5 milliliter eend orph tube. Centrifuge the tubes at 800 G for five minutes. Following removal of the S supernatant, make a hole in the lid of each tube.
Using a push pin, add 100 microliters of minus his medium to the pellet of one tube. To select a haplos to the PT of the other tube, add 100 microliters of minus loo medium. To select alpha haplos.
Rotate the tubes at 30 degrees Celsius overnight. The final optical density at 600 nanometers should be less than 0.5. If optical density at 600 nanometers tends to be too high triol during at room temperature.
To induce GFP, add 100 microliters of fresh minus hiss or minus LU medium containing 20 micrograms per milliliter, doxycycline to the different strains. The final concentration of doxycycline is 10 micrograms per milliliter. Vortex the tube and rotate at 30 degrees Celsius for two days.
To begin flow cytometry, add 900 microliters of pre-filtered TE buffer to a five milliliter polypropylene tube with the cap swapped for a cell strainer cap from a five milliliter polystyrene tube. Polypropylene tubes are suitable for flow cytometry. The strainer cap is desired for removing large particles.
Gently place 75 microliters of the buffer on the cell strainer cap. Add 25 microliters of the induced cells to the solution on the cap while pressing the tip of the perpe and against the mesh. Shoot all of the combined solution through the strainer.
Press down the strainer cap and vortex the tube. Prepare the collection tubes 1.6 milliliter fendor tubes filled with 200 microliters of minus hiss or minus lube medium. Start the cell sorter and adjust settings according to the manufacturer's manual.
Vortex the collection tubes to coat the wall with the medium. Also vortex the tube containing cells before loading them onto the cell sorter. Acquire flow cytometry data of the sample.
A strain without GFP can be a negative control. Draw gates for sorting to avoid cell aggregates, which could exhibit high GFP intensity. Discard outliers with disproportionately large pulse width or area for forward scatter abbreviated FSC using a plot of pulse width and pulse height, or a plot of FSC area and FSC height.
Since large FSC is expected for cell aggregates only take cells in the lower 20%in FSC. Avoid regions with the lowest values for FSC and side scatter or SSC where cell debris is often found. SSC and GFP signal are often positively correlated.
To avoid enriching the cells with a higher SSC value, draw a gate along the periphery of the population using a plot of GFP intensity and SSC to take a similar percentage of the brightest cells from each point along the SSC axis altogether. 0.1 to 1%of the subpopulation selected using FSC and SSC gates before this step should be marked for sorting sort 200 into the collection tube. Vortex the collection tube to cover the sorted cells with medium plate.
A subset of cells on A-Y-P-D-A plate for genotyping. Incubate the plate at 30 degrees Celsius for two days following incubation. Make a lysate for approximately 12 colonies.
Following the steps outlined in the written protocol for genotyping. Analyze one microliter of a diluted lysate using a 10 microliter PCR reaction with a forward primer. That 10 kneels to the upstream flanking region of each deleted gene paired with a common primer that 10 kneels within all deletion loci.
The PCR can be done in a 96 or 3 84. Well format following amplification. Analyze the PCR products using electrophoresis.
Identify haplos likely to have the desired genotypes. Establish these strains on a fresh plate. Also freeze them in 25%glycerol at minus 80 degrees celsius.
Additional tests such as confirmation of the absence of wild type sequences for deleted genes and post field gel electrophoresis are recommended. Repeat sexual cycling with useful strains. Haplo progeny from a cross of two strains were induced to express GFP.
Each gene deletion had a GFP cassette using gates with a flow cytometer, a population of the resulting cells was selected. That is unlikely to contain debris or cell aggregates within this population. The brightest 1%of the cells were sorted and retained by gating cells on the basis of forward scatter area and forward scatter height.
Cell aggregates, which tend to have disproportionately large forward scatter area or excluded by gating cells on the basis of forward scatter and side scatter cells in the lower 20%in forward scatter were accepted while excluding day debris with the lowest forward scatter and side scatter by gating the cells on the basis of side scatter and GFP signal, the more fluorescent cells among those in a given side scatter range were taken to allow comparison of the myotic mix and the negative control sample that does not express GFPA GA identical to the one used for selecting the P three population is shown in the diagram. For the similarly selected cells of the negative control in a separate haplos were analyzed with PCR after they were induced to express GFP and sorted based on fluorescence. The expected number of deletions for an unsorted cell resulting from random segregation was 7.5.
In this particular experiment, the sorted cells had the average deletion number of 8.8 plus or minus 0.3, including five cells containing 10 deletions. DO genotype. The sorted strains PCL was performed using a reverse primer.
That 10 kneels to the downstream flanking region paired with a forward primer. That 10 kneels to the end of the cassette. The presence of a band indicates the presence of the GFP cassette.
After watching this video, you should have a good understanding of how to use the green Monster Method to engineer deletions in yeast.
