Week 1 (7.10-7.16)

1. Saccharomyces cerevisiae S288C resuscitation and subculturing:

1) Prepare liquid YPD media (Solarbio, Beijing, China);

2) Subculture S. cerevisiae S288C from the stock culture;

3) Culture at 30 ℃, 180 rpm.

Notes & Results:

Succeeded. Media became cloudy after 24 hours.

 

2. S. cerevisiae resistance tests:

1) Prepare solid YPD media (Solarbio, Beijing, China);

2) Prepare G418 (100 mg/mL in water) stock solution, sterilize using 0.22 µm filters (BKMAM, Changde, Hunan, China);

3) Prepare solid YPD plates containing 200 µg/mL, 500 µg/mL, and 700 µg/mL G418;

4) Spread 100 µL liquid culture from Step 1 on new plates;

5) Culture at 30 ℃, observe colony growth.

Notes & Results:

It took two days for the colonies to appear.

200 µg/mL G418 was enough to inhibit the growth of S. cerevisiae S288C.

 

 

3. S. cerevisiae genomic DNA extraction:

1) Collect S. cerevisiae from liquid culture by centrifugation at 3000 ×g for 10 min;

2) Use the GeneJet Genomic DNA Purification Kit (Thermo Fisher, Waltham, MA, USA) to extract genomic DNA;

3) Measure the purity and concentration of the DNA samples with a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA).

Notes & Results:

Succeeded. Confirmed by Step 4.

 

4. PCR amplification of ADE4, RIB1, and RIB7:

1) 50 μL PCR: 19 μL deionized water, 25 μL Phusion High-Fidelity PCR Master Mix (Thermo Fisher, Waltham, MA, USA), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), and 2 μL genomic DNA template;

2) The PCR program:

a) one cycle of 98°C for 30s;

b) 30 cycles of 98°C for 10s, 56°C for 30s, and 72°C for 2 min;

c) one cycle of 72°C for 5 min;

3) Run 1% gel electrophoresis to verify the size of amplified fragments;

4) Extract DNA using the TIANgel Midi Purification Kit (Tiangen, Beijing, China).

Notes & Results:

Succeeded.

 

 

Week 2 (7.17-7.23)-Week 3 (7.24-7.30)

5. Recombinant vector construction:

1) Digest pCEV-G4-Km with FastDigest NheI and BamHI (Thermo Fisher, Waltham, MA, USA);

2) Run 1% gel electrophoresis to verify the size of amplified fragments;

3) Extract DNA using the TIANgel Midi Purification Kit (Tiangen, Beijing, China);

4) Measure the purity and concentration of the DNA samples with a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);

5) Use Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, Jiangsu, China);

6) Recombinant vectors:

a) pCEV-G4-ADE4;

b) pCEV-G4-RIB1;

c) pCEV-G4-RIB7;

d) pCEV-G4-RIB1-ADE4;

e) pCEV-G4-RIB7-ADE4;

f) pCEV-G4-RIB1-RIB7;

g) pCEV-G4-RIB1-RIB7-ADE4;

5) Transform the recombinant vectors into Escherichia coli DH5α;

6) Screen on LB plates containing 100 μg/mL;

7) Culture the positive transformants in liquid LB;

8) Extract plasmids using the TIANprep Rapid Mini Plasmid Kit (Tiangen, Beijing, China);

9) Confirm by DNA sequencing (GENEWIZ, Suzhou, Jiangsu, China).

Notes & Results:

We managed to construct pCEV-G4-ADE4, pCEV-G4-RIB1, pCEV-G4-RIB7, pCEV-G4-RIB1-ADE4, pCEV-G4-RIB7-ADE4, and pCEV-G4-RIB1-RIB7. However, when we tried to use pCEV-G4-RIB7-PTV-ADE4-Km as the template to amplify RIB7-PTV-ADE4 by PCR, gel electrophoresis showed no bands. We had to adjust the PCR program to get the fragment we needed.

 

After several failed PCRs, we tried touchdown PCR from 68°C to 52°C. Although the gel electrophoresis results were still smeared, bands at around 2400 bp were identifiable. The fragments were successfully collected through proper gel extraction, enabling the construction of pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km.

 

 

Week 4 (7.31-8.6)

6. S. cerevisiae transformation:

1) Culture S. cerevisiae overnight in 20 mL liquid YPD media at 30°C, 180 rpm;

2) Centrifuge 1 mL culture at 10000 ×g for 30s, discard the supernatant;

3) Wash with 1 mL ddH₂O, centrifuge at 10000 ×g for 30s, discard the supernatant;

4) Prepare 1 mL 1.1 × TE-LiAc solution;

5) Resuspend cells with 100 μL prechilled 1.1 × TE-LiAc;

6) Prepare 10 mL PEG4000 solution: 40% PEG4000 (w/v) in 1 × TE-LiAc;

4) Prepare the transformation mixture: 600 μL PEG4000 solution, 10 μL salmon sperm DNA solution (Solarbio, Beijing, China), and 1 μg plasmid;

7) Add the prepared yeast cells from Step 5 to the transformation mixture;

8) Water bath at 30℃ for 45 min, invert every 10 min;

9) Water bath at 42℃ for 20 min, invert every 10min;

10) Centrifuge at 700 ×g for 5 min, and discard the supernatant;

11) Add 1 mL 2 × liquid YPD media, and incubate at 30℃, 180 rpm for 1h;

12) Centrifuge at 700 ×g for 5 min, and discard the supernatant;

13) Resuspend with 100 μL sterile water, and spread on solid YDP plates containing 200ug/ml G418;

14) Culture at 30℃ for three days;

15) Perform colony PCR to ensure positive transformants.

Notes & Results:

Succeeded.

When doing colony PCR for S. cerevisiae, boiling the cells for 10 minutes before running the program could increase the accuracy of the results.

 

 

Week 5 (8.7-8.13)

7. Measurement of engineered S. cerevisiae growth and riboflavin production in liquid YPD:

1) Prepare 150 mL liquid YPD media (Solarbio, Beijing, China) in each flask;

2) Inoculate the original S. cerevisiae S288C and seven engineered strains into flasks;

3) Culture at 30 ℃, 180 rpm;

4) On hours 0, 8, 24, 32, 48, 56, and 72, measure the OD600s using a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);

5) On hours 0, 4, 8, 24, 28, 32, 48, 52, 56, and 72, take 10 mL from each flask;

6) Centrifuge at 700 ×g for 5 min, and collect the supernatants;

7) Measure the riboflavin concentrations in the supernatants using high-performance liquid chromatography (HPLC) according to GB 5009.85—2016, National Standard of the People’s Republic of China.

Notes & Results:

Results showed that all the strains had a similar growth rate.

Results showed that co-overexpressing ADE4, RIB1, and RIB7 led to the highest amount of vitamin B2 production in S. cerevisiae in liquid YPD.

 

Week 5 (8.7-8.13)-Week 6 (8.14-8.18)

8. Measurement of riboflavin production by engineered S. cerevisiae in steamed buns:

1) Prepare 50 g bread flour (Xinliang Flour, Xinxiang, Henan, China) for each S. cerevisiae strain;

2) Mix the remaining 50 mL liquid culture from Step 7 and the flour;

3) Knead the bread dough for twenty minutes until the surface becomes smooth;

4) Place the dough in plastic bags, and place the bags in a constant temperature incubator at 30 °C for 4h;

5) Use a food steamer to steam the dough for 15 minutes;

6) Measure the riboflavin concentration in the steamed buns using high-performance liquid chromatography (HPLC) according to GB 5009.85—2016, National Standard of the People’s Republic of China.

Notes & Results:

Results showed that co-overexpressing ADE4, RIB1, and RIB7 led to the highest amount of vitamin B2 production in S. cerevisiae in liquid YPD.