Team:USTC

Notebook

Week 1 7.6-7.12

1. Strains po1f and w29 were cultured.

2. PCR for TEF and linearized pUC-19.

3. PCR for coding sequences of each enzyme.

Week 2 7.13-7.19

1. Construction of pUC19 plasmid containing TEF.

2. Replace the TEF coding region with the coding region of each enzyme except ERG8 and BbTPS.

3. PCR for select tags URA3 and LEU4, and verify their function in Y.lipolytica.

Week 3 7.20-7.26

1. Replace the TEF coding region with the coding region of ERG8 and BbTPS.

2. Try to construct double-gene plasmids by ClonExpressⅡ One Step Cloning Kit.

Week 4 7.27-8.2

1. Change the promoter and terminator of ERG10, ERG20, ERG19 and NADH-HMG to GAPDH.

2. PCR for A1, R1 and A3, and connect them by overlap extension PCR.

Week 5 8.3-8.9

1. Construct double-gene plasmids by restricting endonuclease digestion and DNA ligation.

2. Construct HUH select tag by connecting HisG, URA and another HisG.

Week 6 8.10-8.16

1. Finish the construction of all the double-gene plasmids.

2. Construct URA knockout box.

3. Transform the first double gene plasmid into po1f.

Week 7 8.17-8.23

1. Try to knock out URA select tag in po1f.

2. Optimize the culture conditions and transform methods of Y.lipolytica.

Week 8 8.24-8.30

1. Test the result of URA knockout by colony PCR

2. Knock out URA for the second time.

Week 9 8.31-9.6

1. Begin to construct triple-gene plasmid and quadri-gene plasmid.

2. Transform the second double-gene plasmid into the strain.

Week 10 9.7-9.13

1. Knock out URA

2. Optimize the screening method after the knockout.

Week 11 9.14-9.20

1. Construct ACL and ICL.

2. Test the four genes transformed into the strain by colony PCR.

3. Optimize the methods of colony PCR

Week 12 9.21-9.27

1. Finish the construction of triple-gene plasmid and quadri-gene plasmid.

2. Transform triple-gene plasmid into the strain.

3. Test the genes transformed into the strain by colony PCR.

Week 13 9.28-10.4

1. Transform quadri-gene plasmid into the strain.

2. Test all the genes transformed into the strain by colony PCR.

3. Ferment to produce borneol and measure the yield by GC-MS.

Week 14 10.5-10.11

1. Final preparation for iGEM.