Experiments
1. Prepare competent yeast cells
1.1 Prepare yeast seed solution. (Saccharomyces cerevisiae)
Add 1% Angel yeast (AQ-pgaA）into a 4mL test tube with liquid YPD medium.
Mix the solution.
Place the yeast seed solution into a shaker to incubate.
1.2 Dilute the yeast solution by a factor of 10, and measure the OD (optical density at the wavelength of 600nm) using a spectrophotometer in order to determine the titer in cell culture.
Pipette 100μL of yeast seed solution into 900μL sterile water
1.3 Repeatedly centrifuge the cells, remove the supernatant with a micropipette, and pipette sterile water to re-suspend the cell pellet.
1.4 Pipette sterilized cryoprotectant into each test tube of cell pellet in the ratio of 1 to 1.
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Content of yeast cryoprotectant(10mL) :
1) Glycerin 500μL (5%)
2) DMSO 1000μL (10%)
3) Sterile water 8500μL
1.5 Aliquot the solution into 10 tubes, preserve 8 of them into -20°C fridge, and use the other 2 to transfer plasmids with Cas9 gene into competent yeast cells
2. Proliferate plasmids with Cas9 gene and plasmid with sgRNA gene
2.1 Add Cas9 plasmid into Escherichia coli
2.2 Add gRNA plasmid into Escherichia coli
2.3 Place the 2 tubes of solution into the water bath at 42°C for 20 minutes
2.4 Inoculate Escherichia coli with Cas9 plasmids and gRNA plasmids into LB medium with AMP antibiotics.
Take 100μL solution from each tube and centrifuge the solution. After that, take out 70μL supernatant. Apply the remaining 30μL E.coli solution to LB medium evenly.
Place the 2 LB medium into the thermostatic incubator for 8 hours at 37℃
2.5 Prepare 2 tubes of bacterial solution
Mix 4mL liquid LB medium with 4mL AMP antibiotics solution for each tube.
Transfer a single E.coli bacterium with Cas9 plasmids into 1 tube, and gRNA plasmids into the other.
2.6 Place the 2 tubes into a shaker and incubate at 37℃
2.7 Extract Cas9 plasmids DNA and gRNA plasmids DNA from the 2 tubes using the column extraction method.
Discard the flow-through in the column.
Add 500μL washing buffer to the column and centrifuge at 12,000 for 1 minute.
Repeat the washing procedure.
Discard the flow-through in the tube and centrifuge at 12,000rpm for an additional 2 minutes to remove any residual wash solution.
Add 65 °C ddH2O (distilled water) to the tube and centrifuge at 12,000rpm for 1 minute.
Collect the DNA fluid (Cas9 plasmid DNA, gRNA plasmid DNA) in the tube and discard the EZ-10.
3.Transform plasmids with Cas9 gene into competent yeast cells
3.1 Pipette the following reagents into the 2 tubes of cell pellet in the listed order
Cas9 plasmid 2.5μL
Sterile water 31.5μL
PEG3350 240μL (sticky liquid that protects DNA from LiAC)
ssDNA 50μL (single strand DNA, extracted from tuna, which brings Cas9 plasmid into yeast cells. ssDNA should be heated at 100℃ for 10min and be placed into ice immediately before added into the solution.)
LiAC 36μL (catalyst)
3.2 Incubate 2 tubes of cell pellet into a water bath for 20 minutes at 42℃, allowing Cas9 plasmid to go into the yeast cells.
3.3 Centrifuge the 2 tubes of cell pellet and remove the supernatant
3.4 Pipette 500μL of liquid YPD medium into the 2 tubes.
3.5 Prepare YPD-Nourse medium to cultivating the Cas9 yeast
YP medium (solid) 45mL
Glucose 5mL
Nourse (200mg/mL) 50μL
20mg Nourse powder, 100μL sterile water
3.6 Pour the medium into the Petri dishes
3.7 Coat the 2 tubes of solution onto the Petri dishes evenly
3.8 Incubate the Petri dishes into the thermostatic incubator at 30°C
4. Prepare competent yeast cells that contain Cas9 plasmid.
4.1 Prepare yeast seed solution that contains Cas9 plasmid
Add 4mL YPD medium liquid and 2μL Nourse antibiotic. Pick up a single bacterium which has grown for two days in the 30°C incubator and add it into the solution.
Place the yeast solution into the shaker and incubate for 2-3 days.
4.2 Dilute the yeast solution by the factor of 10, and test the OD (optical density at the wavelength of 600nm) using a spectrophotometer.
4.3 Centrifuge the yeast solution at 4500rpm for 4 minutes, and remove the supernatant.
4.4 Add 45mL of sterile water to the yeast solution, centrifuge it at 4500rpm for 4 minutes, and remove the supernatant. (Repeat this step twice)
4.5 Add 400μL of cryoprotectant to the yeast solution.
4.6 Aliquot the yeast solution to ten 1.5ml test tubes.
4.7 Preserve the test tubes in a -20°C refrigerator.
5. Transform plasmids with gRNA gene and homologous recombination template into competent yeast cells that contain Cas9 plasmid.
5.1 Prepare 3 samples (upstream and downstream homology templates + PgaA gene)
PgaA sample
-Add the following reagents in descending order of volume (except sterile water)
a. 16μL sterile water
b. Fx Buffer 25μL
c. PgaA foward primer and PgaA reverse primer 10μL each
d. Diluted ten times dNTP solution 5μL
e. KOD Fx 1μL
f. PgaA template 2μL
Upstream homology template
-Add the following reagents in descending order of volume (except sterile water)
a. 16μL sterile water
b. Fx Buffer 25μL
c. upstream foward primer and upstream reverse primer 10μL each
d. Diluted ten times dNTP solution 5μL
e. KOD Fx 1μL
g. upstream homology template 1μL
Downstream homology template
-Add the following reagents in descending order of volume (except sterile water)
a. 16μL sterile water
b. Fx Buffer 25μL
c. downstream forward primer and downstream reverse primer 10μL each
d. Diluted ten times dNTP solution 5μL
e. KOD Fx 1μL
f. downstream homology template μL
5.2 Place the 3 samples in PCR apparatus
5.3 After PCR, add the following reagents into the 3 samples
-For electrophoresis
Obtain 50μL of three samples relatively, and add the following reagents to each sample:
a. 60% loading buffer 10μL
b. 100% Gelred 6μL
5.4 Prepare agarose gel (2% concentratation), and place it into the horizontal agarose nucleic acid electrophoresis tank
-Agarose 2g
-TAE buffer 200mL
5.5 Pipette 10μL of each solution into each well on the agarose gel.
5.6 After 30 minutes, cut the part of the agarose with fluorescent labelling.
5.7 Extract the DNA from the agarose gel of pgaA and upstream and downstream arm templates using the column extraction method. (slightly different from the plasmid DNA extraction method)
-Measure the weight of the cut agarose gel.
-Add in buffer 2, which is 3 times the weight of the cut agarose gel.
-Incubate the mixture in the water bath at 50°C for 5-10 minutes.
-Move the liquified agarose gel to an absorption column, centrifuge and remove the supernatant.
-Meanwhile, mix absolute alcohol with wash buffer, and add 500μL of the mixture into the same absorption column. Centrifuge the absorption column for 30 seconds, and remove the supernatant. (Repeat 2 times)
-Centrifuge the absorption column for 1 minute.
-Place the absorption column in a 1.5ml collection tube.
-Add 20μL sterile water at 65°C.
-Centrifuge the solution, and preserve the collection tube, which contains the DNA of the PCR products.
-Repeat the above step 3 times.
5.8 Use overlap PCR to integrate the 3 pieces of DNA together into a homologous recombination template.
-Mix the following reagents together.
-PCR setting:
-During the last 5 minutes of the 68 degree Celsius step, add 2μL of primers HR-R and primer HR-L into the tube.
-Then, continue PCR with this setting:
-Preserve the integrated homologous recombination template.
5.9 Insertion of the gRNA plasmid DNA and the homologous recombination template into the yeast competent cells that contain the Cas9 plasmid.
-Mix the following reagents in the listed order;
-Sterile water 24.5μL
-Homologous recombination template 4.5μL
-gRNA plasmid DNA 5μL
-PEG3350 240μL (sticky liquid that protects DNA from LiAc)
-LiAc 36μL (Catalyst)
-ssDNA 50μL (single strand DNA, extracted from tuna, which brings the Cas9 plasmid into the yeast cells. ssDNA should be heated at 100℃ for 10min and be placed into ice immediately before added into the solution.)
-Incubate the mixture in the water bath at 42°C for 20 minutes.
5.10 Centrifuge the solution and remove the supernatant.
5.11 Add 1000μL liquid YPD medium to the solution, and place it in the shaker to incubate at 30°C for 1 hour to revive the yeast.
5.12 Centrifuge the solution and remove 800μL of the supernatant.
5.13 Coat the remaining concentrated solution onto 3 double-antibiotics mediums (Nourse+Hygromycin)
-Prepare 3 double-antibiotics medium:
-50mL liquid YPD medium
-50μL of Nourse mother liquor (200mg/mL)
-100μL of Hygromycin B mother liquor (400mg/mL)
5.14 Wait 2-3 days for the yeast to grow.
5.14 Prepare 4 tubes of yeast solution (2 tubes for one of the medium because the yeast is growing well) that contains Cas9 plasmid, gRNA plasmid, and the homologous recombination template.
-Liquid YPD medium 4mL
-Nourse mother liquor 2μL
-Hygromycin B mother liquor 4μL
-A single yeast from one of the 3 mediums.
5.15 Place the 4 tubes of yeast solution into the shaker to incubate for 2-3 days.
6. Enzyme Activity Test
6.1 Test the OD of the 4 tubes of yeast solution
-OD results: (Dilute the 4 tubes of yeast solution by pipetting 100 μL of yeast solution into 900 μL of sterile water beforehand)
-Solution 1: 0.42 x 10 = 4.2
-Solution 2: 1.10 x 10 = 11
-Solution 3: 1.14 x 10 = 11.4
-Solution 4: 1.41 x 10 = 14.1
6.2 Preserve the 4 tubes of bacterial solution by adding glycerol.
-Add 900 μL yeast solution with 900μL glycerol solution (40%)
6.3 Make 5 double-layer pectin medium (3 for the examination of congo Congo red dye, 2 for the examination for bromophenol blue dye)
6.3.1 Compose Prepare 5 double-layer pectin medium.
-Pour in the first layer of the pectin medium, and wait for the liquid to solidify.
-Inversely stab 7 pipette tips (for 2.5μL pipette) into half the depth of the solidifieds pectin layer.
-Pour in the second layer of the pectin medium, and wait for the liquid to solidify.
-Slowly remove the 7 pipette tips without damaging the pectin layers.
(-repeat the above steps for 3 times to compose prepare 3 double-layer pectin medium for the examination with congo Congo red dye, which is added at the end)
(-add 0.5g bromophenol blue dye into 100mL liquid pectin medium, and repeat the above steps for 2 times to prepare 2 double-layer pectin medium for the examination with bromophenol blue dye)
(use the diluted yeast solution 1, 2, 3 for the examination of Congo red dye, only use the diluted yeast solution 3 for the examination for bromophenol blue dye.)
(-repeat the above steps for 3 times to prepare 3 double-layer pectin medium for the examination with Congo red dye, which is added at the end)
6.4 Inoculate the yeast solution at different concentrations into each hole.
(We only used the first 3 tubes of yeast solution, because the fourth one is not enough for inoculation)
6.4.1 Prepare a control group with Angel yeast, and dilute each of the 3 tubes of yeast solution into 6 different concentrations.
6.5 Pipette 15μL of the 8 prepared liquid into 3 double-layer pectin mediums.
6.6 Pipette 60μL of the 8 prepared liquid into 2 double-layer pectin mediums.
7. Induction Experiment
7.1 Inoculate AQ-pgaA glycerol bacteria into 10ml YPD medium, culture at 30℃, 220r/ml shaking for 36h, and detect the cell concentration OD600=0.6295;
7.2 Take 5ml of bacterial solution in a 50ml centrifuge tube, centrifuge at 1500g at 4℃ for 5min, and collect the bacterial pellet;
7.3 Take 1ml of YPD induction medium containing 2% galactose to fully suspend the bacteria, and supplement the induction medium to 50ml, and culture all the bacteria in a 250ml Erlenmeyer flask (1:50 inoculation), 30℃, 220r/ml Shock training
8. DNS Test
8.1 Detection principle: pectinase hydrolyzes pectin β-galacturonic acid, and reacts with dinitrosalicylic acid (DNS) to form a purple-red compound. The color intensity of this compound is proportional to the concentration of galacturonic acid. The absorbance is measured by a spectrophotometer, and the pectinase activity in the sample is calculated by comparing with the standard curve.
8.2 Pectinase extraction: 48h sample is not broken, the supernatant is not centrifuged, and the supernatant is centrifuged after crushing;
8.3 Dilution of standard pectinase: Take an appropriate amount of standard pectinase (1mg/ml) and dilute:
8.4 Adding samples: add them in the order of the solutions in the table below to avoid bubbles and mix carefully;
8.5 Determination of DNS: Take 1ml pectin standard solution and add it to a 15ml centrifuge tube, add 2ml DNS reagent, boil water bath for 5min, cool tap water to room temperature, take 1ml into a cuvette, and measure OD value at 540nm. Take the absorbance value as the ordinate and each standard concentration (mg/ml) as the abscissa to draw the pectin standard curve.
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