Notebook

Dec.2012
IGEM-SCUT team was established. Every members brainstormed and presented their own ideas, then we discussed the feasibility of these ideas.

Jan.2013 to Feb.2013
After consulting our professors, the final project named E.cerevisiae was determined. Our team was divided into three parts, group of gas generating, odorant sensing and modeling. What gave us confidence was that our project was approved by the dean of the school of bioscience & bioengineering in our university (South China University of Technology). What’s more, we had a training camp at our school to learn some theoretical knowledges.

March.2013 to April.2013
Most of us were the first time to do experiments, so we had another training to improve our skills. And we have applied a lab for our project successfully.

April.2013 to present
Experiment started…

April.2013
WEEK 1
At the begining of our experiments,we got to make a plenty of preparation, which included the preparation of the culture medium and the activation of bacterium .

WEEK 2
We still made some preparation for our experiments, in the meantime,we attended our classes. We were enthusiastic to the experiments although most of us had done few experiments. This week,we learned to prepare competent cells of Escherichia Coli (Top 10) and started to cultivating bacterium (Lactobacillus and BL21) using MRS and LB medium. We also did transformation experiments using plasmid and the newly-preparation Top-10 competent cellS.

WEEK 3
We made abstraction of the genome of Lactococcus lactis using the reagents that prepared by ourselves. Meanwhile, we transformed plasmid YEP352 into competent cells(E.coli). We also learned to prepare competent cells of BL21. To amplifiedα-Als gene,we did a PCR experiment.We did authentication of plasmid YEP352 by using enzyme E.coRⅠ.

May.2013
WEEK 1
We made preparation of YPD and FS medium that cultivated saccharomyces cerevisiaea and Vibrio fischeri. We used electrophoresis to examinate the enzyme-digested plasmid. And we had constructed the system of diaceytl. What’s more, we cultivated the Vibrio fischeri into FS medium.

WEEK 2
We learned to do colony PCR to examine whether the target gene had transformed into the bacterium or not. We also used two kinds of restriction enzyme digestion to examine. Abstraction of the genome of Vibrio fischeri was done and the concentration of the genome was measured.

WEEK 3
To cultivate Bacillus thuringiensis, we prepare some ICPM medium. We did electrophoresis to identify the genome of the Vibrio fischer. When the Bacillus thuringiensis had grown,we abstracted its genome.We transformed the PET-22b(+) into competent cells of BL21. We searched the method of examining the diaceytl.(preparation of MR-VP medium)

Oscillating Odorant
July and August
WEEK 1
PET-226(+)-αALS was successfully tested by restriction digest enzyme. We also sent it to be sequenced. We used IPTG to induce the PET-226(+)-αALS, and the result from the SDS gel electrophoresis showed that we had successfully constructed the system of diaceytl generating.

WEEK 2
We constructed system A:C0060(aiiA+LVA), B:LuxI+LVA, 2:C0062, 5:pluxR, 6:pSB3C5, 7:Q04400(tetR), 8:Psb4a5, 10:GFP.

WEEK 3
aiiA+LVA, LuxI+LVA,pLuxI, pLuxR were successfully authenticated by restriction enzymes digestion, but there was a bad news that restriction digestion of tetR was failed. We used IPTG to induce the PET-226(+)-αALS, and used standard liquid of diaceytl to react with OPD to get the standard curve. To make sure PET-226(+)-αALS wound not react with peptone during the process of growth, we used the Mq medium. We made a big fault that constructed three wrong systems those days, which because we misunderstood the name of pLuxI,luxR and some genes.

WEEK 4
We used the Biobrick C[K54600], D[I13263],E[K546001] to construct system C+pLuxI and E+Psb3c5.

WEEK 5
We finished the CⅢ construction.

WEEK 6 and 7
We combined C with K2，P5 with K1，SS+SⅢ+ pLuxI+R+Αals+2T.

September to October
WEEK 1
We had constructed a standard Ndh curve.

WEEK 2
Gradient quenching PCR of PET-22b(PT).
Construction of C-Ⅲ+K and CⅢ+PSBⅠK3 .
Authentication of systems of CⅢ+K, CⅢ+PSBⅠK3 by restriction enzyme.
Observation of the fluorescence in systems of CⅢ, CⅢ+SⅢ, CⅢ+K3, CⅢ+PSBⅠK3.
We observed that the systems of CⅢ+SⅢ,CⅢ+K3,CⅢ+PSBⅠK3 showed the fluorescence successfully.
Our immobilization was not quite successful, which the bacterium were scorched.
We made the ligation of PT and IP using SolutionⅠ.
We measured the concentration of ndh in systems of C, C+K, CⅢ , CⅢ+K.
We also measured the intensity of fluorescence , which we found all the systems were OK.
Except the CⅢ+k.

WEEK 3
We found that system CⅢ+SⅢ and PT did not work.We got to pick new colony to cultivate.
We begin making preparation of biobrick dry powder.

Odarant sensing
May
WEEK 4
To amplified the odr-10 and rho,we did PCR.Then we used it to do electrophoresis of which products were retrieved the day after.

July and August
WEEK 1
The third time to construct the system of rho-odr10.

WEEK 2
Rho was successfully amplified by PCR, and we got the transformant of flag-rho-odr10.

WEEK 3
We began to constructing the systems of Ⅰ:rho-flag-odr10, Ⅱ:flag-rho-odr10, Ⅲ:EcoRⅠ-XbaⅠ-rho-odr10-SpeⅠ-PstⅠ, and we got the good news that Ⅱ was finished through the restriction digestion.

WEEK 4
According to the cataphoresis we got the results that flag-odr10 was successfully amplificated by the #10rho-odr10 and #23rho-odr10.

WEEK 5
SⅠsystem was constructed successfully.
We constructed Ⅳ:rho-GFP and it worked.

September
WEEK 1
Authentication of the construction of reporter fus1-BFP-ADH1 was successful.
What is more, we accomplished the construction of the two systems of pGAL1+Flag+Odr-10+GFP+ADH1+Fus1+BFP reporter+ADH1 and pGAL1+Flag+Brho+Odr-10+GFP+ADH1 +Fus1+BFP reporter+ADH1

WEEK 2
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 3
We strove to complete our projects’ wiki and practice the presentation .

WEEK 4
As the competition approaching,we made some preparation for setting out.