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:
Gal1+Hbd+GFP+ADH1
Gal1+Crt+GFP+ADH1
Gal1+Ccr+GFP+ADH1
Gal1+AdhE2+GFP+ADH1
Gal1+cox6+Erg10+GFP+ADH1
Gal1+cox6+Hbd+GFP+ADH1
Gal1+cox6+Crt+GFP+ADH1
Gal1+cox6+Ccr+GFP+ADH1
Gal1+cox6+AdhE2+GFP+ADH1
Gal1+Erg10+cyc1
Gal1+Hbd+ADH1
Gal1+Crt+cyc1
Gal1+Ccr+ADH1
Gal1+AdhE2+cyc1
Gal1+cox6+Erg10+cyc1
Gal1+cox6+Hbd+ADH1
Gal1+cox6+Crt+cyc1
Gal1+cox6+Ccr+ADH1
Gal1+cox6+AdhE2+cyc1
Promoter:GAL1. Terminater:Cyc1.ADH1

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. A try 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.

CO2 fixed simulation lab

May

Week 1
We successfully prepared our TOP10 strain to be competent. We started to do the PCR in order to get our targeted gene such as GFP and leading peptide. And meanwhile, we had to practice our lab skill and improve our abilities.

Week 2
We developed the cooperative contract with the Genewiz that helped us synthesize the scaffold protein GBD, PDZ and SH3 with the ligand behind them. Also, this week we made our plan and time table.

Week 3
We successfully constructed the YFP, BFP, GFP and OFP with the RFC 23.At the same time , we waited for our gene from Genewiz.

Week 4
We got the leading peptide Tom22 by PCR and added it into the plasmid yeplac181. We decided to add it behind the GBD, SH3, PDZ fusion until they was finished synthesis.

June
Because of the final exam was coming. We must bury our heart in preparing the exam. So the experiment was stopped. Besides, we got the synthesis gene GBD, SH3, PDZ from Genewiz.

July

Week 1
We still reviewed our courses and hoped to get the good GPA. The last exam would be held in 7th, July. After that, we would concentrate on the experiment.
Week 2
This week we carried on our experiment. Unfortunately, we were puzzled by the contaminated strains but we could not find out why our strains contaminated. So first of all ,we cleaned our super clean bench but it didn't work. At the same time ,we continued our experiment, but we failed all the experiment and could not get any results. That's so crazy. So we continued checking all the factors which resulted in contamination. We remade our LB plates and check our competence cell. But there're still the contaminated phenomena. We still did some microscopic examination for our strains. But it was normal. How should we do?

Week 3
We still could not construct anything. We asked our instructor to give us the new strain. But the contaminated phenomena was still here. We talked it with our advisor and they didn't know what on earth it's going on. So this week we also do nothing.
Week 4
This week our antibiotic Amp had been consumed. So we remade them and found out that the contaminated phenomena went to the end. This week we sped up constructing our device. This week we built the parts including:
PRK
Rubisco
CA
BFP+GBD ligand
YFP+SH3 ligand
GFP+PDZ ligand in Yeplac 181

August

Week 1
Lucky, we did not face any problem so we added the promoter GAL1 in front of the parts mentioned above. And then, the construction of scaffold protein started.
Week 2
The work we done to construct the device was:
1.PCR or cut it down from plasmid
2.Cut it with four restriction enzymes
3.Link them with ligase.
4.Transform
5.Pick up the E. coli and shake it with LB.
6.Obtain the plasmid and have a check.
So this week we almost constructed all the parts.

Week 3
We successfully constructed the GAL1+BFP+GBD ligand+ADH1, GAL1+GFP+PDZ ligand+ADH1, GAL1+YFP+SH3 ligand+ADH1, GAL1+PRK+GBD-ligand+ADH1,TDH3+RuBisCo+SH3-ligand+ADH1,TEF2+GroEL+ADH1,TEF2+GroES+ADH1,TDH3+CA+PDZ-ligand+ADH1,TEF2+GFP+ADH1,TDH3+GFP+ADH1,GAL1+GFP+ADH1. And the optimizing of scaffold protein had been finished a half of all. All of them were
GAL1+GBD+SH3+PDZ+Tom22+ADH1
GAL1+GBD+SH3+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+PDZ+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+PDZ+PDZ+Tom22+ADH1
GAL1+GBD+SH3+PDZ+ADH1
GAL1+GBD+SH3+PDZ+PDZ+ADH1
GAL1+GBD+SH3+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+PDZ+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+PDZ+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+PDZ+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+PDZ+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+PDZ+ADH1
GAL1+GBD+SH3+SH3+SH3+SH3+PDZ+PDZ+PDZ+PDZ+ADH1

Week 4
Oh, this week we got the bad news as if we were struck by lightning. We forgot one thing, if we wanted to construct the fusion protein, we must delete the stop codon TAA. But we forgot this work. So we must design the new primers to do the fusion PCR to mutate the DNA, and added them to the plasmid.

September

Week 1
This week new term started. Because of the stop codon problem, we reconstructed the device. Some of them was finished. But the parts of PRK, CA, RuBiSco met some problems.
Week 2
Some of our parts started to be made the biobricks. Such as the GAL1+BFP+GBD ligand+ADH1, GAL1+GFP+PDZ ligand+ADH1, GAL1+YFP+SH3 ligand+ADH1, and GAL1+BFP+GBD ligand+ADH1+GAL1+GFP+PDZ ligand+ADH1+GAL1+YFP+SH3 ligand+ADH1, but in the scaffold protein respect, we couldn't combine the Tom22 + ADH1 with scaffold protein parts.

Week 3
This week we partly constructed the 1:1:1 scale and other scale scaffold protein plasmid in yeplac181. So there's no doubt that we should transmit it into yeast with the bind ligand added the fluorescent protein so that we could check whether the ligand worked. After the cultivation, we used Confocal Laser Scanning Microscope to get our results.

Week 4
We continued the work last week, and constructed the other scales between GBD,SH3 and PDZ protein. Also, we started to made the growth curve of our wild yeast as well as the one containing the plasmid. We spent all the night and some member got cold.

October

Week 1
We started to prepare our wiki, posters and the presentation. But our experiments still went on .We changed our thoughts about the design of wiki, poster and our uniforms.

Week 2
We sped up composing our wiki, and drew some figures to express our ideas. On the other side, we carried on the construction of the device and planed to checked the carbon capture pathway we built by GC-MS.