Team:SCUT/Team/Notebook

Butanol lab notes

Construction of the n-butanol pathway Members in charge of this part: Fan Chuyao, Hu Weipeng Introduciton: To build up the n-butanol pathway, we have constructed the parts as follow: 2014.5~2014.6 Contents: learning of molecular construction, PCR techniques, transformation of plasmid, several kinds of RFC and their theory and roles in molecular construction. Attempt to construct COXVI+GFP+ADH1 on pSB1C3
5.20~5.31
Contents:Obtain COXVI from yeast genome by PCR
1.Digest Vector and COXVI
2.Assemble COXVI to the pSB1C3 with pSB1C3 on it.
3.Verification
Note:failed for the first time.
7.7~7.24
Note: We have a great touble in plasmid DNA extractiobn which makes a serious effect on our schedule. Finally we find that the cause of the problem is the invalid of ampicillin.
7.25~8.31
Contents: moloecular construction
Note:After we figured out the question we met before, we continued our schedule smoothly. Since our enzyme is assembled in plasmid pUC57,which is provided by Genwiz, and GFP+ADH1 is assembled in plasmid pSB1C3, we digestd GFP+ADH1 from pSB1C3 and assmbled it in pUC57 after the enzyme.We first used pUC57 as our vecto, and then we transported our parts to yeplac181(hbd,crt,erg10) and yeplac352(ccr,adhe2)
　　However, new obstables will always come unless we stop moving on.
　　Some parts length is very close to the vector, it’s very hard to verify whether we succeed or not. We overcome this problem by assemble the parts in other vector whose length is much longer.
　　We have touble in constructing the mitochondrial targeted n-butanol pathway.COXVI has failed to link with enzyme for so many times. We think the cause might be the short length of COXVI(120) and the much larger size of the vector.In the end ,we choose to obtain GAL1+COXVI from the molecule GAL1+COXVI+GFP+ADH1 that as been sequenced rightly by PCR. Then we assemble GAL1-COXVI in our vector and we do it. After that, we continued to assemble the enzymes after GAL1-COXVI.This time the thing become much more easier and we do have a great success.
　　Sometimes we would encounter some phenonmenon that although we had verified the sequence length by double enzyme digestion,the sequencing results did not match the reference sequence.
　　We find the sequence of Erg10 has more than one cleavages,which lead to a result that we can’t get the right sequence before gel extraction, so we have to mutate the cleavage so that it can’r be digested by fast digest enzyme.
　　We find a serious problem that our enzymes have termination codon TAA ,which means our enzymes cannot fuse with the GFP., so we have to knock down TAA by fusion PCR.
9.1~10.1 Contents： Knock down the termination codon by fusion PCR. Notes: We have two round of PCR to get a whole mutated sequenceAt first, we use PrimerStar( TAKARA) because of running out of KOD FX, it works in the first round, however, it fails in the second round. We have desperated for a time. The situation become optimistic when we buy new KOD FX and finally, we succeed to complete the mutation job. Finish molecular construction. Notes:After constructing the indivial parts,we need to assemble Gal1-erg10-cyc1,Gal1-hbd-ADH1,Gal1-crt-CYC1 in yeplac181, which is leucine auxotroph and Gal1-Ccr-ADH1,Gal1-Adhe2-Cyc1 in yeplac352,which is uracil auxotroph. Here we get stuck again. We have failed for many times but can’t figure out the problem. Futhermore, our competent cells are contaminated, ampicillin also has some problem. Even though we refresh the ampicillin and buy competent cells, we still can’t get the job done. Construct biobricks. Notes: Luckily, this part goes on smoothly. Proceed inducible expression. Notes: The procedures of inducible expression. 1.transform plasmid into yeast by electrophoretic transfer 2.incubate at 30℃ for two days. 3.pick up a colony from the plate and transfer to YNB-glucose mixed growth medium, incubate in the shaker for 24 hours 4.Wash off the glucose by PBS and incubate with galactose for 12 hours. 5.Analyze by fluorescence microscopy. To verify the COXVI, we also use Mito Tracker to stain mitochondria and do an overlap with the green fluorescence. Conclusion: No matter what we get at last, we improve ourselves a lot and gain the friendship. We share joy and pain , laughs and tears, we are SCUT-iGEMers.