Experiment 5: Gene Knockouts

 

Overview:

 

You will be performing a Gene Knock-out experiment in Aspergillus nidulans.  You will be deleting the UBC11 gene and replacing it with a selectable marker (the Af pyrG gene).  You will accomplish this by first, constructing a linear DNA fragment that is homologous to the DNA regions that flank the UBC11 gene, which contains the Aspergillus fumigatus pyrG gene in place of UBC11. 

            You’ll construct this DNA molecule by “fusion PCR” using the strategy diagramed in the top, left figure on the last page of this handout.  A related strategy can be used to “epitope-tag” the gene (shown in the top right figure, same page).  The rationale for using this strategy is to generate constructs suitable for gene deletions (aka knockouts) or tagging (knock-ins) rapidly without having to clone them.  This approach has been used primarily in yeast but has recently been adapted for filamentous fungi (Yang et al., 2004) 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 (the Af pyrG gene, in the class).  PCR amplification of all three fragments as templeate (5’ flanking region, Af pyrG, 3’ flanking region) using 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 deletion construct will be transformed into A. nidulans, where it will replace the endogenous UBC11 locus by homologous recombination (middle figure, last page), resulting in a deletion of UBC11 and an insertion of the Af pyrG gene in it’s place.  Selection for Af pyrG (growth in the absence of uracil) will identify transformants that contain the PCR fusion product integrated somewhere in the genome.  PCR will be used to screen for those transformants in which homologous recombination occurred (bottom figure).  Note that all transformants will yield a PCR product using the 5’ For and 3’ Rev primers that amplify the Af pyrG gene, whereas only transformants generated by homologous recombination at the UBC11 locus will yield products with the primer combinations, P1 + 3’ Rev or 5’ For + P6.

            A key reagent in this procedure is the A. nidulans strain called TNO2A7, which has a mutation in a gene (nkuA) required for Non-Homologous End-Joining (NHEJ).  NHEJ is the major pathway used by most eukaryotic cells to repair dsDNA breaks and appears to be the major pathway by which transforming DNA gets inserted into a chromosome.  The NHEJ pathway also acts on exogenous DNA added to cells during transformation, and inserts the DNA fragments randomly into the genome, making the frequency of insertion by homologous recombination low.  In the absence of NHEJ, A. nidulans now acts like yeast and uses primarily homologous recombination.  Thus, one can screen a few transformants by PCR with confidence that most/all of them will be gene knockouts/knockins (Nayak et al., 2006).  Mutation of NHEJ genes has been shown to be a generally applicable in filamentous fungi to increase the frequency of homologous recombination and might be applicable to higher organisms as well. 

 

Details:

 

1.  Generate PCR products corresponding to 5’ and 3’ flanking regions of UBC11 using the Expand Long PCR system.  The TAs will provide you with three PCR tubes; 10X Buffer 1; 10 mM dNTPs; A. nidulans genomic DNA; oligonucleotides P1, P3, P4 and P6, and the Af pyrG Cassette.  Set up two PCR reactions as follows:

                                                5’ Flank PCR Reaction             3’ Flank PCR Reaction

            ddH2O                                    38 ul                                                     38 ul

            10X Buffer 1                            5 ul                                                       5 ul

            10 mM dNTPs                         2 ul                                                       2 ul

            P1 primer                                 1 ul                                                       XXXX

            P3 primer                                 1 ul                                                       XXXX

            P4 primer                                 XXXX                                                 1 ul

            P6 primer                                 XXXX                                                 1 ul

            Genomic DNA                         2 ul                                                       2 ul

            subtotal                                    49 ul                                                     49 ul

 

 

Give the tubes to the TA who will add 1 ul ELT Enzyme and run the PCR reaction using the conditions: Cycle 1 94 deg 2 min; Cycle 2 94 deg 10 s, 50 deg 30s, 68 deg 2 min, repeat 10 times; Cycle 3 94 deg 15 s, 50 deg 30 s, 68 deg 2 min, increasing by 20 s each repeat, repeat 20 times; Cycle 4 68 deg 7 min; Cycle 5 4 deg, ∞.  Save your buffers, primers, genomic DNA, cassette, dNTPs at – 20 degrees.  Save the third PCR tube for the next period.

 

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

            a) Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.

            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.

 

Keep purified PCR products on ice or in the freezer.  Determine the concentration of each PCR product by measuring absorbance at 260 nm and the quality of the product by running 5 ul on a gel (5 ul PCR product, 4 ul ddH2O, 1 ul 10X loading buffer) along with DNA markers.  Load your gel first and then give your PCR products to the TAs who will measure absorbance at 260 nm and give you a printout of the results.

 

3.  Set up Fusion PCR reactions as follows (you have what is needed saved from the previous lab period):

 

                        ddH2O                        ____ ul (= 36.5 – (5’ flank volume + 3’ flank volume)

                        10X Buffer 1                5 ul

                        10 mM dNTPs             2 ul

                        P2 primer                     1 ul

                        P5 primer                     1 ul

                        5’ flank                         ____ ul (100 ng)

                        3’ flank                         ____ ul (100 ng)

                        Af pyrG Cassette          3.5 ul

                        subtotal                        49 ul

                       

Give hand tube to TA who will add enzyme and run the PCR reaction using the conditions: Cycle 1 94 deg 2 min; Cycle 2 94 deg 10 s, 50 deg 30s, 68 deg 4 min, repeat 10 times; Cycle 3 94 deg 15 s, 50 deg 30 s, 68 deg 4 min, increasing by 20 s each repeat, repeat 20 times; Cycle 4 68 deg 7 min; Cycle 5 4 deg, ∞.

 

4.  THE TAs will DO THIS STEP and HAVE YOUR FUSION PRODUCT READY FOR YOU BY THE NEXT LAB PERIOD.  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 quality of each PCR product by running 5 ul on a gel along with DNA markers (5 ul PCR product, 4 ul ddH2O, 1 ul 10X loading buffer).  When your gel is running, give your PCR products to the TAs who will determine the concentration of DNA by measuring absorbance at 260 nm.  They will give you a printout of the results.

 

5.  Transform Fusion PCR product into A. nidulans. 

 

            a)  100 ul of cells in a sterile, 2 ml, snap cap tubes will be provided to you by the TAs.  Keep the tube on ice.  Each tube of 100 ul cells is to be used in one transformation.  You will be doing only one transformation today.

 

            b)  Add 10 ul of your fusion PCR product to the cells.  Mix gently by re-pipetting 5 times. 

 

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

 

            d)  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.

 

            e) Plate the transformation solution onto four plates.  For each plate, pipet 270 ul of the transformation mix into 3 ml molten Aspergillus top-agar medium lacking uracil (YG KCl).  [Note to TAs: Prepare 4 tubes of 3 ml top agar medium per group plus 8 tubes for me and have ready in 50 degree water baths.]  Immediately mix by vortexing vigorously for 5 seconds and pour the entire contents of the tube onto one petri plate containing medium lacking uracil (YG KCl).  Plate four plates with 270 ul each (most of the transformation mixture will be used up).  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.

 

            f) 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.  The TA will make stocks of 3 of your A. nidulans transformants and grow cells that you will use to isolate genomic DNA during the next lab period.  The Aspergillus transformants will be called (group#)T-1,-2, and -3 (for example, group1T-1; group1T-2; group1T-3).

 

6.  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 7 μl of RNase A stock solution (100 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 10 min at 65°C. Mix 2–3 times during incubation by inverting tube.  This step lyses the cells.

            d) Let the tube cool some, 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.  

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

            e) Apply the lysate to the QIAshredder Mini Spin Column (lilac) placed in a 2 ml collection tube and centrifuge for 2 min at high speed.

            f) Transfer flow-through fraction from step 4 to a new 1.5 ml tube (not supplied) without disturbing the cell-debris pellet.  Typically 450 μl of lysate is recovered. If less than 450 ul is recovered, check with the TAs on what to do next.

            g) Add 1.5 volumes of Buffer AP3/E to the cleared lysate and mix by pipetting.  To 450 μl lysate, add 675 μl Buffer AP3/E and mix immediately.  Reduce the amount of Buffer AP3/E accordingly if less lysate is recovered. A precipitate may form after the addition of ethanol but this will not affect the DNeasy procedure.  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* and collection tube.

            j) Place DNeasy Mini Spin Column in a new 2 ml collection tube (supplied), 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 2 min at high speed 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 collection tube. Note: Following the spin, remove the DNeasy Mini Spin Column from the collection tube carefully so the column does not come into contact with the flow-through, as this will result in carryover of ethanol.

            l) Transfer the DNeasy Mini Spin Column to a 1.5 ml microcentrifuge tube and 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.

            j) Quantitate your DNA yield by measuring absorance at 260 nm using the NanoDrop device and running 5 ul on a gel with MW markers (5 ul DNA + 4 ul ddH20 + 1 ul 10X loading buffer).

 

7.  Perform control and experimental PCR reactions to determine whether your transformants carry a deletion of UBC11 using the Sigma JumpStart Taq PCR system.  You’ll be provided with primers 5’ forward for Af pyrG Cassette and 3’ reverse for Af pyrG Cassette; 10X Buffer.  You already have primer P6, dNTPs, and your genomic DNA from your transformants.  Set up the PCR reactions as follows:

 

                                    Control reaction (one per transformant)             Experimental Reaction (one per transformant)

ddH2O                                                ____ ul (41 – vol genomic DNA)                                  ____ ul (41 – vol genomic DNA)

10X Buffer                                           5 ul                                                                               5 ul

10 mM dNTPs                                     1 ul                                                                               1 ul

5’ forward Cassette                              1 ul                                                                               1 ul

3’ reverse Cassette                               1 ul                                                                               XXXX

P6                                                        XXXX                                                                         1 ul

Genomic DNA                         ____ ul (100 ng)                                                           ____ ul (100 ng)

subtotal                                                49 ul                                                                             49 ul

 

 

Give the tubes to your TA and he will add 1 ul enzyme and set up the PCR reaction, using the conditions: Cycle 1 94 deg 2 min; Cycle 2 94 deg 30 sec, 50 deg 30 sec; 72 deg 4.5 min, repeat 30 times; Cycle 3 72 deg 10 min; Cycle 4 4 deg, ∞. 

 

8.  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 4 ul ddH2O and 1 ul 10X loading dye.  Run MW markers on either end of the gel.  Analyze the results (What should be present in each lane of every transformant?  What should be present if the transformant was generated by the desired homologous recombination event?)  Record your group’s results on the blackboard along with those of the other groups.

 

9.  Record and Discuss Results

            Did any of your PCR reaction give unexpected results?  Provide a hypothesis to explain why that might have happened.  What percentage of all Aspergillus transformants in the class were deleted for UBC11?  Is deletion of UBC11 a lethal mutation or not?  Explain.

 

DNAs.

 

UBC11 oligonucleotides P1, P2, P3, P4, P5 and P6. Each at 15 pmole/ul.  Asprgillus genomic DNA, at ~ 100 ng/ul.  Genomic DNA from the transformants you generate.

 

PCR Reagents:

 

10mM dNTP mix

10x Expand Long Buffer

ELT enzyme

10x JumpStart PCR Buffer

JumpStart enzyme