Protocols

I. Single fragment plasmid construction

1. Target gene synthesis

Primers: ylPOT1_F/ylPOT1_R,ylMFE1_F/ylMFE1_R,ylPOX1_F/ylPOX1_R,ylPOX2_F/ylPOX2_R, ylPOX3_F/ylPOX3_R, ylPOX4_F/ylPOX4_R,ylPOX5_F/ylPOX5_R and ylPOX6_F/ylPOX6_R.

Amplify gene ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 and ylPOX6 usingY. lipolytica genome as template and purify the PCR products.

2. Skeleton acquisition

Double digest the plasmid pYLXP' with enzyme SnaBI/KpnI for 3h and purify.

3. Gibson assembly

Prepare Gibson assembly system: 1μL skeleton + 3μL target gene fragment + 4μL Gibson assembly enzyme, 50°C, 1h.

4. Transformation of E. coli competent cells.

Put the assembled product into E. coli competent cell DH5α, incubate on ice for 30min, heat shock at 42°C for 90s, incubate on ice again for 3min, spread on LB+Amp plate, incubate at 37°C for 12-16h..

5. Colony PCR

Use one single colony on the plate as a template for PCR verification. After picking the spots and expanding the incubation, add to PCR system. Add the correct one to the LB+Amp liquid and incubate for 12-14h.

6. Plasmid extraction

Extract the plasmids with the plasmid kit.

7. Verification by restriction digestion

Cut the plasmid into fragments of different length for verification after finding the suitable restriction site, then send correct plasmid for testing.

II. Multi-fragment plasmid construction

1. Double digest the plasmid pYLXP'-ylMFE1- ylPOT1 Double digest t NotI/Nhel for 3h, and then purify to obtain PTEF-ylMFE1-TXPR2-PTEF-ylPOT1-TXPR2 expression box.

2. Skeleton acquisition (pYLXP'-ylPOX1 as an example)

Double digest the plasmid pYLXP’-ylPOX1 with Not I/Nhe I for 3h, and purify to obtain the plasmid skeleton pYLXP’-ylPOX1_Not I/Nhe I containing the gene ylPOX1 expression box.

3. T4 ligase assembly

Assemble the PTEF-ylMFE1-TXPR2-PTEF-ylPOT1-TXPR2 expression box and the plasmid skeleton pYLXP'-ylPOX1_NotI/NheI with T4 ligase, 16°C for 12h [assembly system: 1μL T4 ligase+1μL T4 buffer+2.5μL skeleton ( large fragment) + 5.5μL expression box (small fragment)].

4. Transformation of E. coli competent cells

5. Colony PCR

6. Plasmid extraction

7. Enzyme digestion verification

III. Yeast po1f transformation (pYLXP’-ylPOX1 as sample)

1. Streak yeast po1f on the YPD plate and incubate at 30°C for 36-48h;

2. Pick a single colony on plate and add to YPD liquid medium, incubate for 12h.

3. Transfer po1f into a 2mL centrifugal tube at 12000rpm for 1min, discard the supernatant.

4. Take 5μL of single-stranded DNA, heat at 95°C for 3min, and transfer to centrifuge tubes of step 3 after cooling.

5. Take 5μL lithium acetate and 5μL DTT solution and 500ng corresponding volume of pYLXP’-ylPOX1 plasmid (500ng/measurement concentration) into centrifuge tubes of step 3..

6. Take 80μL of PEG4000, pipette and mix the bacteria, so that the added reagents, plasmids and bacteria are fully mixed, if necessary, shake it in a vortex shaker for 10s.

7. Place the centrifuge tubes in a 37°C water bath for 1h, and shake in a vortex shaker for 10-15 seconds every 10 minutes. After 1h, plate YNB and incubate at 30°C for 48-60h..

Note: plasmid transformation required

IV. Biomass detection (experimental results are shown in the table below)

1. Pick a single colony of the transformant on all the plates in Experiment III and put into 3mL YNB solution and incubate at 30°C for 48h;

2. Add 1mL of seed liquid to 25mL of fermentation broth (the ingredients are as follows), and add to two bottles respectively as a control.

3., Put into a 30°C, 170rpm shaker, ferment for 120h, take samples every 24h, measure OD600 and record the data;.

*Medium components:

V. Oil production experiment (the experimental results are shown in the figure below)

1. According to the results of Experiment IV, transform the more dominant plasmids (pYLXP'-ylPOT1-ylMFE1-ylPOX4 and pYLXP'-ylPOT1-ylMFE1-ylPOX5) and the control group (pYLXP') into Y. lipolytica po1f-Z5.

2. Pick a single colony of the transformant grown on the plate and connect it to the seed liquid and fermentation liquid.

3. Put it in a shaker at 30°C, 170rpm, and ferment for 96h.

4. Centrifuge and freeze-dry the sample, and perform methyl esterification treatment (find methyl esterification method below);

5. Carry out gas chromatography to identify the type of oil produced.

Methyl esterification method:

1. Measure the OD600 of the fermentation broth and record the data, confirm the sampling standard OD600 value is 5, determine the addition volume according to the different OD600 values of the samples, centrifuge at 12000 rpm for 2min, and discard the supernatant.

2. Add 500μL of 1mmol NaOH-methanol solution, mix with the precipitation evenly, shake in a small shaker at 1200rpm for 2h.

3. Add 40μL of concentrated sulfuric acid and 400μL of n-hexane in order, shake again at 1200rpm for 10-20min, and centrifuge for 3min;.

4.Take the supernatant through the organic membrane and conduct gas chromatography.

Table 1：Experiment IV-OD600 values

Figure 1：Experiment V-gas chromatography

Appendix

Protocols for plamid contruction

1. Genome DNA extraction

(1) Take 500μL bacterium sample solution and add to 1.5mL microfuge tube.
(2) Centrifuge at 15000RCF for 4 minutes.
(3) Gently remove the supernatant with pipettor.
(4) Add 100μL cell lysis buffer and vortex thoroughly.
(5) Add 300μL pure ethanol, then repeat step 2 and 3.
(6) Add 400μL 75% ethanol, then repeat step 2 and 3.
(7) Incubate with 65°C metal bath for 5~10 minutes.
(8) Add 50μL ddH2O, then centrifuge at 15000RCF for 1 minute.
(9) Store the extract at -20°C.

2. PCR(polymerase chain reaction)

(1) Reaction system:
① ddH2O: 13μL
② Genome DNA
③ TAKARA PrimerSTAR PCR Mix: 15μL
④ Primer-F: 1.3μL
⑤ Primer-R: 1.3μL
(2) Procedure
① Add ①②③ into PCR tubes and mix them thoroughly.
② Add the primers ④⑤.
③ Proceed with the reaction by using a PCR machine with corresponding programme loaded ( pre denaturation: 5 min+95℃; denaturation: 30s+95℃; annealing:15s+53℃; elongation: 1000bp+1min+72℃; number of cycles: 34).
(3) Products are stored in -20°C refrigerator.

3. Electrophoresis

(1) Materials
① Agarose: 5g
② Buffer 1×TAE: 400mL
③ Nucleic acid dye: 20μL
(2) Gel preparation
① Add agarose and buffer TAE together.
② Microwave for 5 minutes. Take out and mix well, and then microwave heat for 2 minutes.
③ Add nucleic acid dye after cooling.
④ Pour into the mold for cooling.
(3) Procedures
① Electrophoresis at 100V for 0.5 to 1 h
② Analyze the electrophoresis results by UV light.

4. DNA purification (recycling)

(1) Add buffer DE-B of 3 times sample volume. Then pour into chromatographic columns after mixing thoroughly.
(2) Centrifuge at 10000RCF for 1 minute.
(3) Add 500μL buffer W1, then repeat step 2.
(4) Add 700μL buffer W2(with pure ethanol), repeat step 2.
(5) Pour out the waste liquid, then repeat step 2.
(6) Add 20μL ddH2O, then repeat step 2.
(7) Measure DNA concentration with Nanodrop.

5. DNA gel recycling

(1) Add 300μL buffer DE-A into microfuge tubes which contains the gel.
(2) Melt the gel in 75°C metal bath for 10 minutes. The solution will become red.
(3) Add 150μL buffer DE-B, and yellow color will be revealed.
(4) Pour the solution into chromatographic columns and put them in 2mL microfuge tubes.
(5) Centrifuge at 10000RCF for 1 minute.
(6) Pour out the waste liquid, add 500μL buffer W1, then repeat step 5.
(7) Pour out the waste liquid, add 700μL buffer W2(with pure ethanol), repeat step 5.
(8) Pour out the waste liquid, add 350μL buffer S3, repeat step 5. Then stand for 2 minutes.
(9) Dissolve DNA fragments at 65°C metal bath for 2 minutes, with 20μL ddH2O added per tube.
(10) Repeat step 5.
(11) Measure DNA concentration with Nanodrop.

6. Enzyme digestion

(1) Reaction system of 10μL:
① ddH2O: 7μL
② Green Buffer: 1μL
③ SnaBI enzyme: 0.25μL
④ Kpnl enzyme: 0.25μL
⑤ Plasmid: 1.5μL
(2) Incubate at 37°C for 30 minutes

7. Gibson assembly

(1) Reaction system of 8μL:
① Linearized vector: 1μL
② Targeted DNA fragments: 3μL
③ Assembly enzyme(/): 4μL
(2) Incubate at 50 °C for 1 hour

8. E.coli transformation

(1) Competent e.coli cell mixture is stored at -80 °C and taken out 10 minutes before plasmid construction is accomplished.
(2) Procedure:
① Incubate the competent cell mixture on ice for 30 minutes.
② Heat shock each transformation tube with a 42°C water bath for 70 seconds. Slightly shake the tubes to make the plasmids better absorbed.
③ Put the transformation tubes back on ice for 3 minutes.
④ Label the bottom of the plate with LB+amp+plasmid name and the date.
⑤ Drip bacterium sample onto the bottom of the plate with 100μL pipettor.
⑥ Gently move a plastic spreader back and forth through the sample across the plate.
⑦ Store in 37°C incubator with the labeled side on top.

9. E.coli plasmid extraction

(1) Add 1.5mL of the stock culture to each 2mL microfuge tube.
(2) Centrifuge in microfuge tubes at 12000RCF for 1 minute.
(3) Pour off the supernatant.
(4) Re-suspend the pellet with 250μL buffer S1 (with RNase added) for each microfuge tube.
(5) Add 250μL of buffer S2 to each tube and invert the tubes 10 times to mix the contents. Then stand the tubes for 1 minute.
(6) Add 350μL of buffer S3 to each tube and mix by inverting 10 times. White precipitate will be formed.
(7) Centrifuge the tubes at 12000RCF for 10 minutes.
(8) Pour the supernatant into chromatographic columns and put the columns in collection tubes(1.5mL microfuge tube).
(9) Centrifuge at 12000RCF for 1 minute.
(10) Pour out the waste liquid, then add 500μL buffer W1 to each tube. Centrifuge at 12000RCF for 1 minute.
(11) Pour out the waste liquid. Add 700μL buffer W2 (with pure ethanol added) and centrifuge at 12000RCF for 1 minute.
(12) Pour out the waste liquid, then centrifuge at the same speed for 1 minute.
(13) Dry the tubes in 65°C metal bath for 1 minute (with 50μL ddH2O added to each tube)
(14) Measure the DNA concentration with Nanodrop. (concentration within ±0.3ng/μL is considered an acceptable value for ddH2O)

10. Inoculation of bacterial culture

(1) Add 2~3mL LB liquid cultural medium and 2μL ampicillin solution into bacteria preserving tube.
(2) Invert the tube containing bacterial culture 5~8 times to mix the contents.
(3) Add 10μL bacterial culture to each tube, mix thoroughly.
(4) Take the tube near the alcohol lamp before closing the lid.
(5) Incubate for 12~15 hours.

11. Colony PCR

(1) Reaction system of 10μL:
① Taq: 5μL
② TEF-F: 0.5μL
③ XPR2-R: 0.5μL
④ ddH2O: 3μL
(2) Procedure:
① Prepare the primer powder into a solution.
② Prepare the reaction system in the PCR tube.
③ Pick the independent single colony onto a new plate with a toothpick, and then insert the toothpick into the PCR tube.
④ Proceed with the reaction by using a PCR machine with corresponding programme loaded.

12. Yeast transformation

(1) Buffer system of 110μL:
① PEG4000 (50%): 90μL
② Lithium acetate: 5μL
③ ssDNA: 5μL
④ DTT: 5μL
⑤ Plasmid: 5μL
(2) Procedure:
① Heat the ssDNA at 95 ℃ for 3 minutes in advance.
② Prepare the buffer system and mix well.
③ 37 ℃ water bath for one hour (take out and mix every 15 minutes).
④ Apply the bacterial solution to the plate.
⑤ Incubate at 30℃

13. LB liquid medium

(1) ddH2O: 1000mL
(2) Tryptone: 10g
(3) Yeast extract: 5g
(4) NaCl: 10g

14. LB solid medium

(1) ddH2O: 1000mL
(2) Tryptone: 10g
(3) Yeast extract: 5g
(4) NaCl: 10g
(5) Agar: 25g

15. YPD liquid medium

(1) ddH2O: 900mL
(2) Tryptone: 20g
(3) Yeast extract: 10g

16. YPD solid medium

(1) ddH2O: 900mL
(2) Tryptone: 20g
(3) Yeast extract: 10g
(4) Agar: 20g

Team:Worldshaper-Nanjing/Protocols