The overall goal of this procedure is to generate precise gene deletion mutants in fungi. This method can help answer key questions in functional genetics of fungi, such as what role a particular gene of interest plays in the ability of a fungus to cause disease. The main advantage of this technique is that it allows very rapid construction of plasmids for generating precise deletions.
Through this method can provide insight into any fungus that has an effective homologous recombination system. It can also potentially be applied to other systems, such as protist or vermilion cells. We first had the idea for this method when we developed the DelsGate system, which is related to Oscar, but is used for protoplast transformation rather than via agrobacterium.
Visual demonstration of this method is critical as there are number of optimized or non-intuitive steps. Demonstrating the procedure will be Nicole Crenshaw, a technician from My Laboratory. This strategy for designing primers requires the genomic region of the gene of interest that includes the open reading frame, and at least two kilobase flanking the gene on each side, in a word-processing file.
Begin by highlighting the open reading frame intended for deletion, and label the start and stop code-ons. Identify and highlight the adjacent open reading frames within the downloaded sequence. Next, use a primer design tool, to design PCR primers for the five prime flanks of the gene of interest open reading frame.
Use the two kilobase five prime of the gene of interest open reading frame to design PCR primer pair O1 and O2 to generate a minimum size produce of one kilobase. Choose the result that places the reverse O2 primer closest to the open reading frame. Make sure the adjacent open reading frame is not impacted.
Capture a screenshot of the assays, and copy and paste the primer sequences into the word processing document. After repeating the process for the three prime flank to generate primers O3 and O4, add appropriate attach sites to the five primands of all four primers. Lastly, design primers to be used for deletion confirmation and confirmation of homologous recombinance, following the directions in the text protocol.
Begin this procedure by setting up two PCR reactions:one for the five-prime flank, and the other for the three-prime flank. Add the following to both reaction mixers:29.5 microliters of sterile distilled water, five microliters of 10x LA PCR buffer, eight microliters of 2.5 millimolar dNTP mix, five microliters of 25 millimolar magnesium chloride, one microliter of template, and 0.5 microliters of hi-fidelity Taq. To the reaction for the the five-prime plank, add 0.5 microliters of Primer O1, and 0.5 microliters of Primer O2.To the reaction for the three-prime flank, add 0.5 microliters of Primer O3, and 0.5 microliters of Primer O4.Perform PCR with the following reaction conditions:94 degrees Celsius for one minute, then 30 cycles of 94 degrees Celsius for 30 seconds, 60 degrees Celsius for 30 seconds, and 72 degrees Celsius for two minutes, and a final step of 72 degrees Celsius for five minutes before holding indefinitely at 10 degrees Celsius.
To determine product size and relative concentration, perform electrophoresis on the PCR products on a 0.8%agar of 1x TLE gel for approximately 125 volt hours in a standard mini-gel apparatus. Post stain the gel with non-ethidium stain according to the manufacturer's instructions, and visualize on a UV illuminator. If the five-prime and three-prime flanks are in roughly even concentrations, combine them.
If they are not of equal concentration, combine the low concentration product with an estimated equal amount of product from the higher-yield reaction. Use an infinity column kit to co-purify the PCR products according to the manufacturer's protocol. Next, carry out the BP clonase reaction by mixing the following:one microliter of the assembly vector, two microliters of the marker vector, one microliter of combined flanks, and one microliter of clonase.
Incubate at 25 degrees Celsius for 16 hours. Terminate the reaction by adding 0.5 microliters of protonase-K, and incubating at 37 degrees Celsius for 10 minutes. After transforming E.coli cells with the BP reaction and isolating DNA as described in the text protocol, identify the correct construct by performing a double digest with Hindiii 3 and KPN 1.
Following identification of the OSCAR deletion construct by double digesting sequencing, the construct is transformed into agrobacterium tumefaciens strand AGL1. The next step is to transform fungi with AGL1 containing the OSCAR deletion construct. Begin by preparing a fungal spore suspension with sterile water at 2 times 10 to the sixth spores per milliliter.
After diluting the spores, place 500 microliters of the fungal spores suspension in a 1.5 milliliter micro-centrifuge tube. Use a blue sterile disposable loop to add an easily visible glob of the deletion plasmid containing AGL1 into the spores suspension. Vortex until the bacterial cells are well dispersed.
Place a cellulose membrane filter on each of four six-centimeter petri dishes containing co-cultivation medium and then add about four sterile glass beads per dish. Pipette 100 microliters of the conidia agra suspension onto the center of each membrane filter, and spread the suspension with beads to cover the whole surface of the membrane filter. After removing the beads, allow the membrane filters to dry in the hood for approximately ten minutes, and then wrap the petri dishes with paraffin film.
Invert the dishes and incubate for two days at room temperature. After two days, transfer each membrane filter onto a six-centimeter asper gel selective medium plate containing Hygromycin B and kanamycin. Incubate at room temperature for five to eight days.
To isolate Hygromycin-resistant colonies, use sterile toothpicks to transfer putative transformants onto six-centimeter potato dexterous agar plates containing Hygromycin and kanamycin. Incubate the plates at room temperature. Once the transformants form colonies on the plates, use a sterile toothpick to transfer a small amount of mycelium or yeast cells from the colony into 100 microliters of lysis solution in a micro-centrifuge tube.
Incubate the mixture at 85 to 90 degrees Celsius for 20 to 30 minutes. Store the crude extract containing genomic DNA at negative 20 degrees Celsius until ready to perform PCR. To identify deletion mutants, first carry out PCR to detect the gene of interest open reading frame and the selectable marker for each transformant.
Use the same reaction conditions for the production of OSCAR constructs. After single spore isolation and thermal lysis, repeat the PCR for the open reading frame and Hygromycin, as well as PCR for detecting homologous recombination of the five-prime and three-prime flanks. Subsequently, run an agarose gel of the PCR products.
The OSCAR method generates a plasmid containing the selectable marker gene, surrounded the flanks of the target gene to be deleted, which is a verticillium dahliae gene in this example. When double digested, the release of an insert of 4, 247 base pairs in this case, confirms the correct plasmid structure in all lanes, except lanes five and 11. A PCR approach is used to confirm the deletion.
This example shows the analysis of five fusarium verticillioides transformants. In the top gel, lanes two to six are the five-prime plank amplifications, and lanes seven to 11 are the open reading frames amplifications. In the bottom gel, lanes two to six are the selectable marker gene amplifications, and lanes seven to 11 are the three-prime flank amplifications.
The presence of bands in the five-prime flank, three-prime flank, and selectable marker gene amplifications and the absence of bands in the open reading frame amplification, confirm that strands 1131, 1132 and 1133 are deletion transformants. The presence of bands in the open reading frame and selectable market genes amplifications, and the absence of bands in the five-prime flank and three-prime flank amplifications, indicate that strands 1134 and 1135 are atopic transformants. Gene deletions can also be confirmed by Southern blot.
Once mastered, this technique can generate Oscar deletion plasmids ready for fungal transformations in two days, not including sequencing time. While attempting this procedure, it's important to remember to use hi-fidelity Taq preliminaries, and to be certain that adjacent genes will not be affected. Following this procedure, other methods to test mutant phenotypes can be performed in order to answer additional questions, like whether a gene is important in particular processes, such as drug sensitivity or resistance.
Don't forget that working with living organisms, such as agra bacterium and fungi can be hazardous, particularly if an individual is amino compromised. And precautions such as hand-washing after handling, should always be performed with this procedure.
