Due to the epidemic, we were unable to return to school for experimental verification. The following are the experimental steps we have sorted out through literature research.
The phage T4 obtained from plaques was heated at 100 °C for 10 min and then cooled down to 8 °C. The inactivated phage T4, which was provided by Prof. Song Gao at Jiangsu Ocean University, was stored at -20 °C and used directly as the template for PCR.
Horizontal electrophoresis tank, electrophoresis apparatus, gel mold and comb (Liuyi, Beijing), microwave (Midea, China), laboratory bottles (250 mL), UVCI-1100 Gel Imaging system (Major Science, US)
Agarose (Sangon, Shanghai), Ethidium bromide (stock concentration of 10 mg/mL, Thermo Fisher, US), 50× TAE Buffer (Thermo Fisher, US), DNA Ladder (0.2-12 kb, 12 bands), (Beyotime, Shanghai)
1) Measure 1 g agarose.
2) Take 20 mL of 5×TAE solution, add water and dilute to 1 L.
3) Mix agarose powder with 100 mL 1×TAE in a flask and microwave it for 1-3 min until the agarose is completely dissolved.
4) Let agarose solution cool down to about 50 °C, about 5 min.
5) Add EtBr to a final concentration of approximately 0.2–0.5 μg/mL.
6) Pour the agarose into a gel tray with the well comb in place.
7) Place newly poured gel at 4 °C for 10-15 min OR let sit at room temperature for 20-30 min, until it has completely solidified.
8) Add loading buffer to each of the DNA samples.
9) Once solidified, place the agarose gel into the gel box.
10) Fill gel box with 1x TAE until the gel is covered.
11) Carefully load a molecular weight ladder into the first lane of the gel.
12) Carefully load your samples into the additional wells of the gel.
13) Run the gel at 80–150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1–1.5 hours, depending on the gel concentration and voltage.
14) Turn OFF the power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
15) Visualize the gel by the UVCI-1100 Gel Imaging system, analyze the size of DNA fragments by comparing them with bands of the DNA ladder.
Microcentrifuge (Thermo Fisher, US), thermostat water bath (Kuansons, China)
DNA agarose Gel, SanPrep Column DNA Gel Extraction Kit (Sangon, China)
1) Cut the gel block containing the target fragment from the agarose gel and weigh it.
2) Add 3–6 volumes Buffer B2 to 1 volume of gel block (100 mg gel ~100 μL), and incubate in a water bath at 50 °C for 5-10 min.
3) Transfer the solution to the adsorption column and centrifuge at 8, 000 × g for 30 s, pour out the liquid in the collection tube.
4) Add 500 μL Wash Solution and centrifuge at 9, 000 × g for 30s. Pour out the liquid in the collection tube.
5) Repeat step 4.
6) Centrifuge the empty adsorption column at 9, 000 × g for 1 min.
7) Put the adsorption column into a clean 1.5 mL centrifuge tube, add 40 μL Elution Buffer to the center of the adsorption membrane, leave it at room temperature for 1 min, and centrifuge for 1 min. Preserve the DNA solution in the tube.
PCR thermocycler (Thermo Fisher, US), Microcentrifuge (Thermo Fisher, US), micropipette (Ependourf), Vortex (Cyto-bio, China), thermostat water bath (Kuansons, China), Incubator (Zollo, China), Agarose gel electrophoresis system (refer to 2 and 3)
pMet-Cas9 and pTrp-gRNA (from Prof. Zhongjun Qin, UCAS), BglII (Thermo fisher, US), dNTP mix (10 mM each in ddH2O), sterile ddH2O, TOPO XL-2 PCR Cloning Kit (Thermo Fisher, US), 1X TAE buffer (40 mM Tris-acetate, pH 8, 1 mM EDTA), Rapid DNA ligation kit (Beyotime, China), sterile ddH2O
37 °C water bath incubation for 4 h.
Reaction system:
Incubate at 25 °C for 30 min.
Table 1. Insert sequence of pTrp-gRNAα and pTrp-gRNAa.
Examining 2 μL of the product by 0.8 % agarose gel electrophoresis. (refer to 2 and 3)
Microcentrifuge (Thermo Fisher, US), triangular flask, Shaker (Zhicheng, China), microplate reader (BioTek, US), Incubator (Zollo, China), flasks, centrifuge tube (Corning, US), Vortex (Cytobio, China)
The highly transformable Saccharomyces cerevisiae strain VL6–48 (MATα, trp1-Δ1, ura3-Δ1, ade2-101, his3-Δ200, lys2, met14) (Bnbio, China)[1], YPD liquid medium (Solarbio, US), Sorbitol Solution (Sangon, China), STC solution (Coolaber, China), YPD medium (Solarbio, US), ddH2O
1) Prepare the premixed solution for transformation
2) Add 360 μL premixed solution to a tube with competent cells, gently mix the precipitation repeatedly to make the yeast cells fully suspended.
3) Incubate the tube in a water bath at 30 °C for 30 min, and mix well per 10 min.
4) Heat the tube in a water bath at 42 °C for 30 minutes and mix well per 10min.
5) Centrifuge for 1 min at 12,000× g. Remove the supernatant.
6) Resuspend the cell with 1 mL YPD Liquid Medium, shake at 30 °C for 60 min. Centrifuge for 1 min at 12,000× g. Remove the supernatant.
7) Add 100 μL sterile water to the precipitation to make it resuspend. Transfer to the corresponding yeast culture plate and spread the cells gently with a bacterial cell spreader. Incubate at room temperature for 10 minutes. After the liquid was absorbed, Inverted the plate was and cultured at 30 °C for 2–4 days.
Microcentrifuge (Thermo Fisher, US), triangular flask, Shaker (Zhicheng, China), thermostat water bath (Kuansons, China), flasks, Incubator (Zollo, China)
Competent S. cerevisiae VL6-48 (yMATα) (Obtained in step 5), YPD liquid medium (Solarbio, US), Yeast Transformation Kit (Coolaber, China), SD-Met-Trp and SD-Met (Biofeng, China), IPTG (Thermo Fisher, US)
1) Use the Yeast Transformation Kit (Coolaber, China) to transfer both pMet-Cas9 and pTrp-gRNAα into half amount of S. cerevisiae VL6-48 MATα. Transfer only the pMet-Cas9 into another half. (See operation instructions on the manual of RK21116 SmaI, ABclonal)
2) Inoculate these samples on SD-Trp-Met medium and SD-Met medium respectively, incubate at 30 °C for 2-4 d. Then yMATa-Cas9Met-gRNATrp and yMATα-Cas9Met will be obtained.
3) Transform all colonies on liquid medium, add appropriate amount of IPTG respectively.
4) Inoculate the sample on SD-Met both, incubate at 30 °C for 2–4 d, then yMATa-Cas9Met and yMATα-Cas9Met will be obtained.
Microcentrifuge (Thermo Fisher, US), micropipette (Ependourf), Vortex (Cyto-bio, China), Agarose gel electrophoresis system (refer to 2 and 3)
Backbone of pRS327, pRS422, pRS423, pRS426 (Biofeng, China), primers for amplification of phage genome fragments (Table 2), SmaI (ABclonal, China), 10× Buffer CutS (ABclonal, China), dNTP mix (10 mM each in ddH2O), sterile ddH2O, TOPO XL-2 PCR Cloning Kit (Thermo Fisher, US), 1× TAE buffer (40 mM Tris-acetate, pH 8, 1 mM EDTA)
Table 2. Primers for linearized vector.
Reaction system:
Incubate at 25 °C for 1 h.
See operation instructions of the manual of SmaI.
Amplify the vector backbone with each primer via PCR.
PCR reaction system:
Use the following cycling parameters for amplicons.
After producing the PCR product, analyze 5–10 µL by agarose gel electrophoresis to verify the size, quality, and quantity of the PCR product. (refer to 2 and 3)
PCR thermocycler (Thermo Fisher, US), Microcentrifuge (Thermo Fisher, US), Agarose gel electrophoresis system (refer to 2 and 3)
Phage genomic DNA (obtained in step 1), Phusion DNA polymerase (Thermo Fisher, US), dNTP mix (10 mM each in ddH2O), TOPO XL-2 PCR Cloning Kit (Thermo Fisher, US), 1× TAE buffer (40 mM Tris-acetate, pH 8, 1 mM EDTA), sterile ddH2O
The primers are on Table 3.
See the operations on TOPO XL-2 PCR Cloning Kit.
Table 3. All primers used in PCR amplify wide-type T4 genome.
After producing the PCR product, analyze 5–10 µL by agarose gel electrophoresis to verify the size, quality, and quantity of the PCR product. (refer to 2 and 3)
Bacterial cell spreader, refrigerated centrifuge (Thermo Fisher, US), thermostat water bath (Kuansons, China), centrifuge tube (Corning, US), Incubator (Zollo, China)
Yeast competent cells (obtained in step 5), Linearized pRS423-SmaI-1-2, pRS426-SmaI-2-3, pRS423-SmaI-3-4, pRS426-SmaI-4-5 (obtained in step 7), phage genome PCR fragments (obtained in step 8), SD-His-Met, SD-Ura-Met (Biofeng, China), PEG 8000 (Solarbio, US), YPD liquid medium (Solarbio, US), Zymolyase 20T (ICN Biomedical, US), Sorbitol Solution (Sangon, China), EDTA (Sigma Chemical, US), Yeast nitrogen base without amino acids (Difco, US), 14 mM beta-mercaptoethanol (ME, Sigma Chemical, US), Taq polymerase (Takara, Japan), Diethylpyrocarbonate (Sigma Chemical, US), Potassium acetate (KAc, Sigma Chemical, US), SDS (Bio-rad, US), Calcium chloride (CaCl2·2H2O), ddH2O
1) Gently mix 200 mL of spheroplast suspension with 2-3 µg of phage DNA fragments and 1 µg of the linearized vector in a 2 mL centrifuge tube. Make 3-5 parallel transformation reactions for each vector. Incubate for 10 min at room temperature.
2) Add 800 µL of PEG 8000 solution into each tube, gently mix by inverting and incubate for 10 min at room temperature.
3) Centrifuge the samples for 5 min at 300-500× g at 5 °C. Remove the supernatant and gently resuspend the spheroplasts in each tube.
4) Incubate the spheroplasts for 40 min at 30 °C without shaking.
5) Transfer the spheroplasts from each transformation reaction into a separate 15 mL Corning tube containing 7 mL of melted SORB-TOP-His selection medium (equilibrated at 50 °C), gently mix and quickly pour agar onto SORB-His plates.
6) Keep the plates at 30 °C for 5-7 d until all the transformants become visible.
7) Transfer 100-300 primary transformants by toothpicks onto SD-His plates lacking histidine. Individually streak 30 colonies onto each master plate.
8) Incubate the plates, each containing a pool of 30 transformants, at 30 °C for 2 d.
9) Replica-plate the master plates on new plates with SD-His-Met and SD-Ura-Met selective medium respectively using replica-plating apparatus and velvet pad. Use the master plates for detection of gene-positive pools
10) Wash out the yeast cells from each master plate with 5 mL of water into 15-mL Corning tubes, and pellet the cells by centrifugation for 5 min at 1,000g at 5 °C. Remove and discard the supernatant.
11) Resuspend each cell pellet in 1 mL of 1 M sorbitol solution by vortex, transfer the suspension to 2-mL Eppendorf microfuge tube and spin for 30 s at 2,000× g at room temperature. Remove and discard the supernatant.
12) Resuspend each cell pellet in 0.5 mL of SPE solution containing ME (1/1,000 dilution), add 20 mL of zymolyase solution into each tube and incubate for 2 h at 30 °C.
13) Harvest the spheroplasts by centrifugation in the Eppendorf microfuge for 5 min at 2,000× g at room temperature and resuspend the pellets in 0.5 mL of EDTA (0.5 M, pH 7.5), mix well.
14) Add 1 mL of diethylpyrocarbonate to each tube at room temperature and vortex well.
15) Lyse the spheroplasts completely by incubation at 70 °C for 15 min.
16) Add 50 mL of 5 M KAc solution to each tube, mix well and leave the tubes on ice for 30 min.
17) Pellet the precipitate by centrifugation for 15 min at 16,000× g at room temperature.
18) Transfer the supernatants to fresh microfuge tubes, fill the tubes with ethanol at room temperature, mix and pellet the DNA by centrifugation for 5 min at 16,000× g at room temperature. Remove the supernatant as much as possible and dry the tubes.
19) Resuspend each damp DNA pellet in 0.4 mL of water.
20) Add 0.5 mL of isopropanol. Mix well and immediately pellet the DNA by centrifugation for 5 min at 16,000× g at room temperature.
21) Remove the supernatant as much as possible and dry the tubes well.
22) Dissolve the final pellet of DNA in 0.3 mL of water.
23) Use 1 µL of the DNA solution in a 50 µl PCR with appropriate diagnostic primers to identify gene-positive pools. Conditions for PCR are not different from the Taq polymerase manufacturer’s suggested protocols. (The primer is on Table 4, no use of primer I and II, II and III, III and IV, IV and I)
24) Touch the streak of each transformant from replica plates with ‘positive pools’ with a sterile disposable tip and then rinse the tip thoroughly in 100 µl mixture of 80 µl water, 20 µl zymolyase solution and 1 µL of ME.
25) Incubate the resulting suspension for 1 h at 30 °C.
26) Add 10 µL of 2% SDS solution. Incubate for 15 min at 70 °C.
27) Add 10 µL of 5 M KAc solution and leave the tubes on ice for 15 min.
28) Spin at 16,000× g for 2 min at room temperature.
29) Transfer the supernatants to new Eppendorf tubes and add equal volume of isopropanol. Precipitate 16,000× g for 2 min at room temperature.
30) Dissolve the pellets in 30 µL of water.
31) Use 1 µL of the DNA solution in a 50 µL PCR with appropriate diagnostic primers to identify gene-positive clones in the pools. (PCR positive colonies include strains yMATa-1His-Cas9Met, yMATα-2Ura-Cas9Met, yMATa-3His-Cas9Met and yMATα-4Ura-Cas9Met)
Table 4. Primers for colony PCR.
Incubator (Zollo, China)
S. cerevisiae VL6-48 (yMATa-1His-Cas9Met, yMATα-2Ura-Cas9Met, yMATa-3His-Cas9Met and yMATα-4Ura-Cas9Met), pTrp-gRNAα and pTrp-gRNAa (Obtained in step 4), pTrp-gRNA-S1S2, pRS422-SmaI and pRS327-SmaI (Obtained in step 7), SD-His-Met-Ura (Biofeng, China), IPTG (Thermo Fisher, US)
1) Spread 500 μL overnight YPD culture on the plate, respectively inoculate yMATa-1His-Cas9Met and yMATα-2Ura-Cas9Met, yMATa-3His-Cas9Met and yMATα-4Ura-Cas9Met on complete YPD agar plates and incubating at 30 °C for 2 d. [3]
2) After incubating at 30 °C overnight, replica the lawns onto SD-His-Ura-Met medium, incubated the plates at 30 °C for 2 d. Colonies forming a patch contain the strain yMATa/MATα-1His-2Ura-Cas9Met and yMATa/MATα-3His-4Ura-Cas9Met that we obtained.
3) Use the Yeast Transformation Kit to transform the linearized pRS422-SmaI-1-3 and pTrp-gRNA-S1S2, the linearized pRS427-SmaI-3-5 backbone and pTrp-gRNA-S1S2 respectively into yeast yMATa/MATα-1His-2Ura-Cas9Met and yMATa/ MATα-3His-4Ura-Cas9Met. Incubate at 37 °C for 1 h.
4) Inoculate the sample on SD-Trp both, incubate at 30 °C for 2–4 d, then y yMATa/MATα-1His-2Ura-Cas9Met-gRNAS1S2Trp and yMATa/MATα-3His-4Ura-Cas9Met-gRNAS1S2Trp will be obtained.
5) Transform all those colonies on liquid medium, add appropriate amount of IPTG respectively.
6) Inoculate the sample on SD-Ade for yMATa/MATα-1His-2Ura-Cas9Met-gRNAS1S2Trp and SD-Lys for yMATa/MATα-3His-4Ura-Cas9Met-gRNAS1S2Trp, incubate at 30 °C for 2–4 d, then yMATa/MATα-12ade-Cas9Met and yMATa/MATα-34lys-Cas9Met will be obtained.
7) Use 1 µL of the DNA solution in a 50 µl PCR with primers I and II (Table 4) for yMATa/MATα-12ade-Cas9Met, and III and IV (Table 4) for yMATa/MATα-34lys-Cas9Met to identify gene-positive pools.
8) Use the Yeast Transformation Kit to transform the pTrp-gRNAα and pTrp-gRNAα are respectively transformed into yeast yMATa/MATα-12ade-Cas9Met and yMATa/MATα-34lys-Cas9Met. Incubate at 37 °C for 1 h.
9) Inoculate the sample on SD-Trp for yMATa/MATα-12ade-Cas9Met and yMATa/MATα-34lys-Cas9Met both, incubate at 30 °C for 2–4 d, then yMATα/MATα-12ade-Cas9Met and yMATa/MATa-34lys-Cas9Met will be obtained.
The sequences of MATa and MATα are on Table 5.
Table 5. Sequence of gene MATa and MATα.
1) Artificial synthesis of MATa and MATα
2) Amplify the sequence via PCR. (step 4)
Table 6. Primer for cloning MATa and MATα.
3) Use the Yeast Transformation Kit to transform MATα and MAT a respectively into yeast yMATα/MATα-12ade-Cas9Met and yMATa/MATa-34lys-Cas9Met. Incubate at 37 °C for 1 h.
Incubator (Zollo, China)
S. cerevisiae VL6-48 (yMATα/MATα-12ade-Cas9Met and yMATa/MATa-34lys-Cas9Met) (Obtained in step 11), pRS426-Smal-1-5 (Obtained in step 7), SD-Ade-Lys, SD-Trp, SD-Ura-Met (Biofeng, China).
1) Spread 500 μL overnight YPD culture on the plate, respectively inoculate yMATα/MATα-12ade-Cas9Met and yMATa/MATa-34lys-Cas9Met on complete YPD agar plates and incubating at 30°C for 2 d.
2) After incubating at 30 °C overnight, replica the lawns onto SD-Ade-Lys medium, incubated the plates at 30°C for 2 d. Colonies forming a patch contain are yMATa/MATa/MATα/MATα-12ade-34lys-Cas9Met we obtained.
3) Use the Yeast Transformation Kit to transform the linearized pRS426-Smal-1-5 and pTrp-gRNA-S1S2 into yMATa/MATa/MATα/MATα-12ade-34lys-Cas9Met. Incubate at 37 °C for 1 h.
4) Inoculate the sample on SD-Trp, incubate at 30 °C for 2–4 d, then yMATa/MATa/MATα/MATα-12ade-34lys-Cas9Met-gRNAS1S2Trp will be obtained.
5) Transform all those colonies on liquid medium, add appropriate amount of IPTG respectively.
6) Inoculate the sample on SD-Ura-Met for yMATa/MATa/MATα/MATα-12ade-34lys-Cas9Met-gRNAS1S2Trp, incubate at 30 °C for 2–4 d, then yMATa/MATa/MATα/MATα-1234ura-Cas9Met will be obtained.
7) Use 1 µL of the DNA solution in a 50 µl PCR with primers I and IV (Table 4) for yMATa/MATa/MATα/MATα-1234ura-Cas9Met to identify gene-positive pools.
Centrifuge (Thermo Fisher, US), thermostat water bath (Kuansons, China)
YeaStar Genomic DNA Kit (Zymo Research, US), absolute ethanol, ddH2O
See operation instructions of D2002 YeaStar Genomic DNA Kit .
Check the quality of the extracted product: Take 2 μL of the product by 0.8 % agarose gel electrophoresis. (refer to 2 and 3)
Gene Pulser Xcell (Bio-rad, US), Centrifuge (Thermo Fisher, US), Incubator (Zollo, China), electric furnace (Midea, China), Cuvette (BTX, US)
Extracted DNA (Obtained in step12), E. coli 10G chemically competent cells (Novo, China), Beef extract peptone medium (Solarbio, US), LB liquid medium (Solarbio, US), Chloroform
1) Extract 3 μL of extracted DNA and electroporate into 20 μL E. coli in a 2 mm gap electroporation cuvette at 2,500 V, 25 μF, 200 Ω using Gene Pulser Xcell.
2) Incubate the cells at 37 °C for 1 h in 1 mL LB liquid medium.
3) Add drops of chloroform to kill the cells and release T4 phage.
4) Prepare the bottom medium (about 10 mL per dish, 1.5% agar), poured onto the plates.
5) Take 0.1 mL of phage suspension, add 0.2 mL of E. coli suspension, mix and add dropwise to the bottom medium.
6) Take the upper medium (0.8% agar), melt and cool to 48 °C, pour 5 mL on the bottom medium, mix well and incubate at 37 °C.
7) Prick a single plaque with an inoculation needle, carefully take the phage, insert it into the liquid medium containing E. coli, and cultivate it at 37 °C. After the bacteria in the tube is completely dissolved, filter and sterilize to obtain purified phage.
Automatic cell counter and counting board (LUNA, Korea), multiskan go microplate (Thermo Fisher, US), 96 Well cell culture cluster (Corning, US), plate, Incubator (Zollo, China)
Transformed E. coli 10 G (Obtained in step 13), LB liquid medium (Solarbio, US), chloroform
1) Incubate the supernatants of transformed E. coli 10 G for 1-2 h.
2) Add chloroform to kill the cells and release phages.
3) Mix the supernatants and pour onto plates, incubate overnight at 37 °C.
4) After incubation, examine each plate and count the number of plaques on each plate that has clearly differentiated plaques.
5) Analyzed for plaque formation and calculate the amount of plaque.
6) Calculate the number of lytic phages per milliliter that were in the original phage suspension using the formula mentioned above.
[1]Larionov, V., Kouprina, N., Solomon, G., Barrett, J. C., & Resnick, M. A. (1997). Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast. Proceedings of the National Academy of Sciences, 94(14), 7384-7387.
[2]Kouprina, N., & Larionov, V. (2008). Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nature protocols, 3(3), 371.
[3]Xie Ze-Xiong, Mitchell Leslie A, Liu Hui-Min, et al. Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae .. 2018, 8(1):173-183.