Our experiment was mainly consisted of five parts: constructing plasmids with different target genes, transforming yeast strains, knocking out the gene PEX10, regulating the culture and fermentation conditions, conducting the quantitative real-time PCR, extracting and detecting the products.
The Microspectrophotometer
The PCR Amplifier
Dry thermostat
Thermostat water bath
The centrifuge
Centrifuge tubes
Preservation tubes
E. coli competent cells (DH5-α)
Bsa I restriction enzyme
LB ampicillin solid medium (100 μg/mL ampicillin)
40% glycerol
DNA Extraction Mini Kit
T4 ligase (T4DLig)
T4 ligase buffer (10× T4 DHA Ligase Buffer)
Our experiment required appropriate enzyme genes and plasmid skeletons. After consulting relevant literature, we designed plasmids on SnapGene software, chose appropriate DNA segments and plasmid skeletons, determined the restriction sites added on the two sides of different target genes, and designed relevant PCR primers.
(1)The total reaction volume of PCR is 25 μl, including 12.5 μL primer STAR max enzyme, 1 μL*2 primers, 0.7 μL template, and 9.8μL double distilled water (ddH2O).
(2)Purification of PCR products and DNA fragments: PCR products and DNA fragments were purified with DNA Extraction Mini Kit (Nanjing Vazyme Biotech Co., Ltd).
①Add the triploid volume of Buffer GL to PCR stock solution, mix with pipette.
②Transfer the solution to the adsorption column, centrifugate the tube at 12000 ×g for 1min, then discard the liquid, and put the adsorption column back into the empty collection tube.
③Add 700 μL Buffer W2 to the adsorption column, centrifugate the tube at 12000 ×g for 1min, and discard the liquid.
④Repeat step ③ once.
⑤Put the adsorption column back into the empty collecting tube and centrifugate it at 12000 ×g for 1min.
⑥Take out the adsorption column, put it in a clean 1.5 ml centrifuge tube, open the cover and heat it at 65℃ for 1 min.
⑦Add 30 μl of ddH2O and centrifuge at 12000 ×g for 1min to get the solution containing the purification of PCR products.
(3)The concentration of target gene or skeleton DNA was measured by microspectrophotometer.
Different target genes were ligated with corresponding promoter/termination/plasmid skeleton at 16℃ using T4 ligase. The recombinant plasmids were transformed in E. coli competent DH5-α, and cultured at 37℃ overnight. The positive transformants were transferred to the LB ampicillin solid medium.
(1)The total volume of the T4-ligation system was 20 μL, including (number of base pairs*0.02/ concentration) ng of plasmid, (number of base pairs *0.04/ concentration) ng of target gene, 2 μL of T4 ligase, ddH2O, and the mixture was bathed at 16℃ for 4-6 h.
(2)The recombinant plasmid was transformed into the competent state of E. coli.
①10 μl mixture of ligation mixture was injected into DH-5α competent which was then placed on ice for 30 min.
②Heat shock under 42 ℃ for 1.5 min.
③Place the competent on ice for 3 minutes.
(3)The E. coli were then spread on LB ampicillin solid medium.
Ampicillin was used to screen the E. coli carried the recombinant plasmids. Monoclonal E. coli grown on LB ampicillin solid medium were selected and then diagnostic PCR was performed to verify the recombinant plasmids. Lastly, the recombinant plasmids were sent to a professional institution for gene sequencing.
(1)Diagnostic PCR
①Total volume of each colony PCR was 10 μl, including 5 μl Taq enzyme, 0.5 μl F and R primers, and 4 μl ddH2O.
②Pick monoclone with sterilized toothpicks into the PCR tube as the templates in the clean bench.
③Mixtures were placed in the PCR Amplifier (Taq enzyme program) and amplified for 30 cycles.
(2)Select an appropriate Marker to indicate the positive band.
(3)Pick and culture the positive colony in LB ampicillin liquid medium for 12h. Extract the plasmids and send to professional institutions for sequencing.
(4)Strain preservation: Mix the E. coli culture liquid having correct sequencing results and 40% glycerol in a 1:1 ratio, and stored in a refrigerator at -80℃.
The Microspectrophotometer
The visible spectrophotometer
Restriction enzymes
DNA Extraction Mini Kit
Centrifuge tube
Dry thermostat
YNB solid medium for fermentation (20 g/L glucose, 5g/L ammonia sulfate, 1.7 g/L YNB without amino acids and ammonium sulfate, 0.4 g/L uracil, or 0.4 g/L L-leucine and 25 g/L agar)
YPD liquid medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose)
Frozen-EZ Yeast Transformation II Kit
Yeast Competent cells were prepared according to the method of Frozen-EZ Yeast Transformation II Kit.
(1)Seeds: Inoculate monoclonal yeast to a conical flask with 10 mL YPD liquid medium and grow under 30℃ at 220 rpm until its OD600 reaching between 0.8-1.
(2)Take 1 mL of the medium into a centrifuge tube in the clean bench, and centrifugate at 4000 ×g for 3min.
(3)Remove the supernatant in the clean bench, add 1mL EZ1 solution, resuspend the strain, and centrifugate at 4000 ×g for 3 min.
(4)Remove the supernatant in the clean bench, add 100 μL EZ2 solution, and resuspend the strain.
(5)Divide the solution into two parts, 50 μl each, and store them in the refrigerator at -80 ℃.
We digested the constructed plasmid to obtain a linear DNA fragment containing target gene, URA/Leu gene and HisG tag, which was then transferred to the yeast competent cells. The engineered Y. lipolytica was obtained by screening.
Since the Yeasts we used were nutrition-deficient (uracil or leucine deficient) strains, once the linear DNA fragment carrying URA/Leu gene was successfully transformed and integrated into Y. lipoltiyca, the transformants could grow on the YNB solid medium. In addition, we placed the HisG tag at both ends of URA gene to recycle the URA gene by adding 5-Fluoroorotic Acid (5-FOA) to the medium to get URA deficient strain allowing for the next round transformation.
(1)Digest the plasmids with the total volume of 50 μl, including 4 μg of plasmids, 2 μL×2 of enzyme, 5 μL of 10X buffer, ddH2O, incubated under 37℃ for 4-5 h.
(2)Agarose gel electrophoresis and purification.
①Cut the gel containing required fragments and then put it into the 2 ml centrifuge tube.
②Add 500 μL Buffer GL and then incubate in the thermostat water bath at 65°C for 4-6 min. Mix the solution every 2-3 min until the gel is completely melted. The following steps are the same in [A-1- (2)]
③Add 700 uL Buffer W2 to the adsorption column, centrifugate the tube at 12,000 ×g for 1min, discard the waste liquid.
④Repeat step ③ once。
⑤Put the adsorption column back into the empty collecting tube and centrifugate the tube at 12,000 ×g for 1 min.
⑥Take out the adsorption column, put it in a clean 1.5 ml centrifuge tube, open the cover and heat it at 65℃ for 1 min.
⑦Add 30 μl of ddH2O and centrifugate the tube at 12000 ×g for 1min to get the solution containing the purified PCR products.
(3)Measure the concentration of target gene or skeleton DNA by microspectrophotometer, and the required concentration should be over 50 ng/μL.
(4)Transform the linearized DNA segment into Y. lipolytica. During the transformation, add 10 μl lineage DNA segment and 500 μL of EZ3 solution into the prepared yeast competent cells, incubate at 28℃ for 1h.
(5)Spread 250 μL of the mix on YNB (including Leu) fermentation solid medium, grow at 30℃ for 48-96 h.
The Microspectrophotometer
The PCR Amplifier
Dry thermostat
The centrifuge
Centrifuge tubes
Preservation tubes
E. coli competent cells (DH5-α)
LB ampicillin solid medium(100 μg/mL ampicillin)
Restriction enzymes
DNA Extraction Mini Kit
YNB solid screening medium
Frozen-EZ Yeast Transformation II Kit
40% glycerol
yeast lysate
100% absolute ethanol
75% absolute ethanol
Set the gene PEX10 homology arm and construct the "gene knockout plasmid", using the same steps as [A].
We transformed the gene PEX10 knockout plasmid into the competent cells of yeast and knocked out the gene PEX10 by homologous recombination.
(1)Prepare the yeast competent cells, the same as [B-1].
(2)Transform gene PEX10 knockout plasmids, the same as [B-2].
(1)Preparation before the experiment
①We randomly selected 12-16 yeast transformers and cultivated them in 5 mL YPD liquid medium at 30℃, 220rpm for 12h (OD600 must under 0.6).
②Preparation of yeast lysate (200mM lithium acetate + 1% SDS solution)
(2)Genome extraction of Y. lipolytica
①Place 500-1000 μL of yeast solution in 1.5mL centrifuge tube, centrifugate the tube at 15000 ×g for 2 min, discard supernatant.
②Add 100 uL of yeast lysate, resuspend the strain, and incubate at 75℃ for 10min, mix the solution at the same time.
③300 uL of 100% absolute ethanol was added to the above solution to precipitate DNA, totally mixed the solution, centrifugate the tube at 15000 ×g for 5min, and then discarded the supernatant.
④400u L 75% ethanol was added, resuspend the strain, centrifugate the tube at 15000 ×g for 5min, and then discarded the supernatant.
⑤The centrifuge tube was placed in a dry thermostat at 65℃ for 10 min to evaporate the residual ethanol.
⑥add 50 μL ddH2O to resuspend the precipitation, centrifugate the tube at 15000×g for 1min. The supernatant was genome DNA of Y. lipolytica.
(3)The PCR system
①Total PCR system was 10 μl, including 5 μl of Taq enzyme, 0.5 μl of F and R primers each, 1uL of template and 3uL of ddH2O.
②The PCR tubules were placed in the PCR Amplifier (Taq enzyme program) and amplified for 30 cycles.
(4)After PCR, we chose appropriate markers, performed agarose gel electrophoresis to whether PEX10 was successfully knocked out.
Conical flask
The visible spectrophotometer
Preservation tubes
40% glycerol
YPD liquid medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose)
YNB solid screening medium (20 g/L glucose, 5g/L ammonia sulfate, 1.7 g/L YNB without amino acids and ammonium sulfate, 0.4 g/L uracil, or 0.4 g/L L-leucine and 25 g/L agar)
YNB fermentation medium (2.5 g/L yeast extract, 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate), 60 g/L glucose)
We inoculated monoclonal yeast on YNB solid screening medium (20-25 monoclones selected at one transformation) into YPD liquid medium, then incubated them at 30℃, 220 rpm for 24h to obtain seed solution.
(1)We obtained seeds as the steps mentioned before and 40% glycerol at a ratio of 1:2 to protect the yeast. We named the yeast in the format of strain-gene-date.
(2)Transfer 500 uL seed medium into 50 mL of YPD fermentation medium, incubated them at 30℃ 220 rpm for 96 h in a conical flask.
The PCR Amplifier
(1)After measuring the extracted yeast genomes described above in [C-3-(2)] by NanoDrop, we calculated and diluted its concentration to 1-3ng /μL,using ddH2O.
(2)We designed the primers for test and reference, using SIGMA online design of qPCR website. In this experiment, we used G6PDH as the reference primer. (Website: http://www.oligoarchitect.com/OligoArchitect/LoginServlet)
Preparation of the qPCR reaction system:10 μL of 2xSYBR qPCR Mix, 0.4 μL of primer F(10 μM), 0.4 μL of primer R(10 μM), 0.4 μL of genome (1-3 ng/μL), and dd H2O up to 25 μL.
(4)PCR program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 20s, 56 ℃ annealing for 20s, and 72℃ for 20s at extension stage. A total of 40 cycles; 56 ℃ temperature gradient rise (1℃ / min) measurement of the melting curve.
(5)The acquired data were calculated according to Eq: C copy number = 2 - (CTTarget-CTReference)
1M sodium hydroxide-methanol solution
Concentrated sulfuric acid
n-hexane
Gas chromatograph
Centrifuge tube
The centrifuge
96-well plate shaker
Vials
The visible spectrophotometer
After fermentation, we extracted the total fatty acids of the engineered Y. lipolytica and detected the content and proportion of various fatty acids with gas chromatograph.
(1)Measure the OD600 of each conical flask by visible spectrophotometer, based on which we calculated the volume of each flask fermentation broth to be inoculated (To final OD600=4). For example, the OD600 of the first conical flask is 8.5, the volume would be 4/8.5=470 uL.
(2)According to the above method, we took the corresponding volume of the fermentation broth from each bottle into different centrifuge tubes, centrifugated at 12,000 ×g for 2 min, and discard the supernatant.
(3)Add 500 μL 1M sodium hydroxide-methanol solution in each tube, mix, and incubate in a 96-well plate shaker for 4-5h.
(4)Add 40 μL concentrated sulfuric acid to each tube, then add 500 μL hexane. Then again incubate in a 96-well plate shaker for 10 min to get fully reacted.Centrifugate at 12000 ×g for 2 min. Then absorbe the upper organic layer and injected it into a vial.
(5)Detected the content of various fatty acids with the Gas chromatograph. In the end, we collected the data and analyzed the results.