Experiment 1, Simple Gene Knockouts

Overview:

You will be performing a Gene Knock-out experiment using Aspergillus nidulans as a model eukaryotic organism. You will be deleting the wA gene and replacing it with a selectable marker (Af pyroA). wA is required for green colony color (green spores): wA mutants are white (white spores). pyroA is required for Aspergillus to grow on medium lacking pyridoxine (vitamin B6). You will accomplish the wA gene knockout by first constructing a gene knock-out construct and then transforming that construct into competent Aspergillus cells and selecting for growth in the absence of pyridoxine. The knock-out construct will be a linear DNA fragment that is homologous to the DNA regions that flank wA, but will contain the Af pyroA gene in place of the gene’s coding sequence. Some of the cells that take up the knock-out construct will insert it into the genome by homologous recombination, which will result in deletion of wA and insertion of Af pyroA in its place.

You’ll construct these DNA molecule by “fusion PCR” using the strategy diagramed in Vector NTI handout for this experiment. This approach has been used primarily in yeast but has recently been adapted for filamentous fungi and could, in principle, be used to generate constructs useful for animal genome manipulations. The strategy for deletions involves generating ~1 kb PCR fragments corresponding to regions that flank the gene of interest. Each flanking fragment has 21 bp sequence at one end that is identical to the sequence at one end of a PCR product that contains a selectable marker. PCR amplification using all three fragments as the template (5’ flanking region, Af pyroA, 3’ flanking region) and primers that hybridize near the ends of the flanking fragments results in formation of a ~ 4 kb amplicon corresponding to the fusion of all three template fragments.

The knock-out construct will be transformed into A. nidulans cells. In Aspergillus cells, like in animal cells, the knock-out construct will have one of three fates: fail to integrate into the genome and be lost, integrate into the genome by non-homologous recombination (NHEJ pathway), or integrate into the genome by homologous recombination. Selection for Af pyroA (growth in the absence of pyridoxine) will identify transformants that contain the PCR fusion product integrated somewhere in the genome. Integration by homologous recombination will result in deletion of wA and an insertion of the Af pyroA gene in its place, and will be detectable as a white colony. PCR will be used to confirm that white transformants are deleted for wA.

Details:

1. Generate PCR products corresponding to 5’ and 3’ flanking regions of wA and using NEBs Phusion, high fidelity PCR enzyme. The TAs will provide you with two PCR tubes containing the reaction mix from NEB, a 1.5 ml tube containing A. nidulans genomic DNA; a 1.5 ml tube containing the Af pyroA Cassette, and 1.5 ml tubes containing a wA del oligonucleotides P1, P2, P3, P4, P5 and P6, Set up two PCR reactions as follows: The 1.5 ml tubes are stock that you will use again later this semester, so save them in your -20 degree box before leaving for the day. Set up two PCR reactions as follows.

5’ Flank PCR Reaction 3’ Flank PCR Reaction

reaction mix in PCR tube 12.5 ul 12.5 ul

ddH2O 10.5 ul 10.5 ul

P1 primer 1 ul XXXX

P3 w/Af pyroA primer 1 ul XXXX

P4 w/ Af pyroA primer XXXX 1 ul

P6 primer XXXX 1 ul

Genomic DNA 1 ul 1 ul

subtotal 25 ul 25 ul

Give the tubes to the TA who will run the PCR reaction using the conditions: Cycle 1 98 deg 2 min; Cycle 2 98 deg 10 s, 65 deg 30s, 72 deg 30 sec, repeat 35 times. Save your buffers, primers, genomic DNA, cassette, at – 20 degrees.

2. When you get you PCR reaction back from the TAs, take 1 ul of each PCR reaction with 9 ul sample buffer and save on ice. You will run this sample along with your purified PCR reaction on a gel to determine the quality of the PCR reaction and the yield of purified PCR product.

3. Purify the products using the Qiagen PCR purification kit.

a) Add 5 volumes of Buffer PBI to 1 volume of the PCR sample in a clean, 1.5 ml tube and mix by vortexing.

b) Place a QIAquick spin column in a provided 2 ml collection tube.

c) To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.

d) Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.

e) To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

f) Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

g) Place QIAquick column in a clean 1.5 ml microcentrifuge tube with the lid cut off.

h) To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 50 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

i) Transfer eluted DNA to a clean, labeled 1.5 ml microfuge tube.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

4. Keep purified PCR products on ice or in the freezer. Determine the concentration of each PCR product by measuring the absorbance using the NanoDrop device (TAs will do this) and by running a gel. Run the 5 ul of the purified PCR product (5 ul purified PCR product, 3 ul ddH2O, 2 ul 5X loading buffer) along with the unpurified sample on a gel (5 ul PCR product, 4 ul ddH2O, 1 ul 10X loading buffer) along with DNA markers.

5. Set up Fusion PCR reactions using NEBs Phusion, high fidelity PCR enzyme as follows (you’ll be given a PCR tube with the reaction mix and you have the other components in your -20 box):

reaction mix in PCR tube 25 ul

ddH2O ____ ul

P2 primer 1 ul

P5 primer 1 ul

5’ flank PCR product ____ ul (100 ng)

3’ flank PCR product ____ ul (100 ng)

Af pyroA Cassette 1 ul

subtotal 50 ul

Give hand tube to TA who will run the PCR reaction using the conditions: Cycle 1 98 deg 2 min; Cycle 2 98 deg 10 s, 65 deg 30s, 72 deg 2 min, repeat 35 times.

6. When you get you PCR reaction back from the TAs, take 1 ul of each PCR reaction with 9 ul sample buffer and save on ice. You will run this sample along with your purified PCR reaction on a gel to determine the quality of the PCR reaction and the yield of purified PCR product.

7. Purify the product using the Qiagen PCR purification kit (protocol above), EXCEPT ELUTE THE DNA USING ONLY 30 ul of EB. Keep purified PCR product on ice or in the freezer. Determine the concentration of each PCR product by running 5 ul of the purified product on a gel along with the unpurified product and DNA markers (5 ul PCR product, 3 ul ddH2O, 2 ul 10X loading buffer).

8. Transform Fusion PCR product into two different A. nidulans strains; MLC1-89 and TN02A7. MLC1-89 is a pyroA4 mutant but is wild type for NHEJ. TN02A7 is a pyroA4 mutant that is also mutant for Ku70, which is thought to be required for NHEJ. [Note to TAs: I will do one of each strain as part of the demo and will transform them using DNA I have or from the class]

a) Take one flask containing Aspergillus protoplasts plus debris for each strain (two flasks total) from the incubator, transfer the contents to a 15 ml conical centrifuge tube. Make sure the volume of the cell suspension in each tube is the same and spin the tubes at 2500 rpm for 10 minutes at 4 degrees (balance the centrifuge using your two tubes - see TAs for help with this).

b) Pour off the supernatant liquid and place the tubes on ice. Note that the majority of the pellet is dark but with a thin, cream-colored layer on top. Gently add 1 ml of WASH buffer, being careful not to resuspend the dark part of the pellet. Use the 1 ml to resuspend the cream colored layer and transfer that to a new 15 ml tube on ice. Add 5 ml more wash buffer, mix gently by shaking. Do the same for the other tube and spin at 2500 rpm for 10 minutes balancing the centrifuge using your two tubes.

c) Repeat the previous step except keep the whole pellet.

d) Repeat the previous step except use RESUSPENSION buffer.

e) Pour off the supernatant liquid, allowing most of the liquid to drain from the tube. Place the tube back on ice and let the liquid in the tube collect at the bottom. Gently resuspend the cells with a P-200 set at 200. If there is at least 200 ul of cell suspension, then you are done. If not, estimate the volume using your pipetman and add enough additional RESUSPENSION buffer to bring the total to > 200 ul. THE GOAL OF THIS STEP IS TO HAVE JUST OVER 200 ul OF WASHED PROTOPLASTS OF EACH STRAIN.

f) Transfer 100 ul of cells of each strain into sterile, LABELED, 1.5 ml microfuge tubes and keep them on ice. Keep the tubes on ice!!! One tube of each strain is to be used as the NO DNA TUBES.

g) Add 10 ul of your fusion PCR product to one tube of each strain (PLUS DNA TUBE). Mix gently by re-pipetting 5 times. Place them back on ice.

h) Add 50 ul of 20% PEG to each tube. Mix gently by re-pipetting 10 times. Incubate on ice for at least 20 minutes (longer is OK).

i) Add 1 ml PEG. Mix by inverting the tube until the solution has coated the whole tube. Incubate at room temperature for 20 minutes, mixing by inverting the tube several times every 5 minutes.

j) Plate the cells in PEG as follows (I will demonstrate this for the class one time):

Pipet 270 ul of the PLUS DNA transformation mix into 3 ml molten top-agar medium lacking pyridoxine (SC minus pyro KCl).

Immediately mix by vortexing vigorously for 5 seconds and pour the entire contents of the tube onto one petri plate containing medium lacking pyridoxine (SC minus pyro KCl).

Repeat this three times for the PLUS DNA TUBES (most of the transformation mixture will be used up).

Pipet 270 of the NO DNA TUBE on one plates using this procedure.

Note to students: don’t pour water bath water into your plates – wipe the water off the outside of the tubes before dumping their contents onto the plate.

[Note to TAs: SC minus pyro should contain uracil and uridine. Prepare 5 tubes of 3 ml top agar medium per group plus 4 tubes for me and have them ready in 50 degree water baths.]

k) Incubate the plates face up on your bench overnight. The TAs will transfer the plates to 37 degree incubators in my lab the next day and will bring the plates to you the following lab session.

9. Observe the results of the transformation. Any colonies on your no DNA control? How many transformants did you obtain? What fraction of the transformants of each strain were white? Select two of the largest, white colonies from the TN02A7 strain and use an inoculating loop to transfer lots of spores to a petri dish containing liquid SC minus pyro medium. The TAs will collect these dishes and incubate them for you and return them to you the next lab period.

[Note to TAs: I’ll do two colonies of each strain from my plates]

10. Collect the mycelium of each of your Transformants using a spatula and the funnel and filter paper. Wash the cells with 200 ml of water and then remove most of the liquid using paper towels (watch demo). Transfer the semi-dry mat of cells to a piece of foil ,wrap the cells in foil and label the foil with your group number, strain name. Place the foil packs in a beaker in the -20 degree freezer. The TAs will lyophilize (freeze-dry) the samples and return a sample to you in the next lab period.

11. Isolate total DNA from each of your transformants using the Qiagen DNAeasy kit. The TAs will provide you with 40 mg of lyophilized cells in a 1.5 ml microfuge tube.

a) Disruption of lyophilised tissue: grind the cells to a fine powder using the dissecting probes provided by the TAs.

b) Add 700 μl of Buffer AP1 and 5 μl of RNase A stock solution (10 mg/ml) to the powder, vortexing vigorously to wet all the powder. You may have to invert the tube and tap it against the lab bench, or use a pipet tip, to dislodge dry, packed down powder that remains dry in the bottom of the tube.

c) Incubate the mixture for 45 min at 65°C. Close the caps if they pop open (they will, maybe more than once). Mix the contents by inverting tube at 15 minute intervals during incubation. This step lyses the cells and dissolves proteins and lipids.

d) Let the tube cool, then spin for 5 minutes at maximum speed in the microcentrifuge. Remove 400 ul of supernatant liquid and transfer it to a new 1.5 ml tube.

e) Add 130 μl of Buffer AP2 to the lysate, mix, and incubate for 5 min on ice. This step precipitates detergent, proteins, and polysaccharides.

f) Spin for 5 minutes at maximum speed in the microcentrifuge to pellet the precipitate. Transfer 450 ul of the supernatant (no debris) to a new tube at room temperature.

g) Add 675 μl of Buffer AP3/E to the cleared lysate and mix by pipetting. Note: It is important to pipet Buffer AP3/E directly onto the cleared lysate and to mix immediately.

h) Apply 650 μl of the mixture from step 6, including any precipitate which may have formed, to the DNeasy Mini Spin Column sitting in a 2 ml collection tube (supplied). Centrifuge for 1 min at high speed and discard flow-through. Reuse the collection tube in step (i).

i) Repeat step (h) with remaining sample. Discard flow-through.

j) Add 500 μl Buffer AW to the DNeasy Mini Spin Column and centrifuge for 1 min at high speed.. Discard flow-through and reuse the collection tube in step (k).

k) Add 500 μl Buffer AW to the DNeasy Mini Spin Column and centrifuge for 1 min at high speed. Discard the flow through. Use the same collection tube to spin the column at high speed for 2 minutes to dry the membrane. It is important to dry the membrane of the DNeasy Mini Spin Column since residual ethanol may interfere with subsequent reactions. This spin ensures that no residual ethanol will be carried over during elution. Discard flow-through and transfer the DNeasy Mini Spin Column to a clean, 1.5 ml microfuge tube that has the lid cut off.

l) Pipet 100 μl of 68 degree Buffer AE directly onto the DNeasy membrane. Incubate for 5 min at room temperature and then centrifuge for 1 min at high speed to elute the DNA. Hot AE is more efficient at eluting DNA from the column, however; room temperature AE will also work.

m) Quantitate your DNA yield by measuring absorbance at 260 nm using the NanoDrop device and running 3 ul on a gel with MW markers (3 ul DNA + 5 ul ddH20 + 2 ul 5X loading buffer).

12. Perform PCR reactions to determine whether your transformants carry a deletion of wA using NEB Phusion PCR reagents. You’ll be provided with 2 PCR tubes containing reaction mix. You will the primers and genomic DNA you just isolated needed to set up the reactions for each genomic DNA sample:

PCR tube with reaction mix 25 ul

ddH2O ____ ul

wA del P1 1 ul

wA del P6 1 ul

Genomic DNA ___ ul (100 ng)

subtotal 25 ul

Give the tubes to your TAs who will set up the PCR reaction, using the conditions: Cycle 1 98 deg 2 min; Cycle 2 94 deg 30 sec, 50 deg 30 sec; 72 deg 4.5 min, repeat 40 times; Cycle 3 72 deg 10 min

9. Analyze the results of the control and experimental PCRs for each transformant by running the reactions on a gel along with MW markers. Mix 5 ul of each PCR reaction with 3 ul ddH2O and 2 ul 5X loading dye. Run MW markers on either end of the gel. Analyze the results.

10. Record and Discuss Results

Document the predicted and actual PCR results, including all photos of gels. Provide a hypothesis to explain differences between predicted and actual results and suggest an experiment to test the hypothesis. What percentage of all Aspergillus transformants were deleted for wA based on colony color (include class data)?

DNAs.

wA del oligonucleotides P1, P2, P3 w/Af pyroA, P4 w/Af pyroA, P5 and P6. Each at 15 pmole/ul (Mirabito lab standard). Aspergillus genomic DNA, at ~ 100 ng/ul.

Other:

Tips, tubes, top agar, plates, gels, DNA markers; access to Nano Drop machine, probes for powdering Aspergillus cells, water baths set to temp, regenerated DNeasy columns, regenerated PCR purification columns (treated with 700 ul 1 N HCl at least overnight and washed 3x with QBT buffer), PB1 buffer, PE buffer (80% ethanol); EB buffer; AP1 buffer (50 mM EDTA pH 8.0, 0.2 % SDS), AP2 buffer (E. coli plasmid miniscreen buffer 3), AP3/E (30 ml of 6 M Guanidine HCl plus 60 ml ethanol), AW buffer (80% ethanol), Spores of strains MLC1-89 and TN02Ay. Protoplasting enzyme, protoplast wash buffer, protoplast resuspension buffer.