Difference between revisions of "Team:Jilin China/Collaborations"

We cooperated with BIT-China this year, a team from Beijing institute of technology in China .and we helped them build a part. So they can test the expression of membrane protein better.

In their project, they mainly want to express C-type GPCR (T1R2＆T1R3) in Saccharomyces cerevisiae. In this process, they are concerned about whether protein can be successfully expressed in the cell membrane , so they want to add BCP（Blue cytochrome protein）to the ORF of T1R2，and they can observe the protein T1R2 on the membrane by fluorescence confocal microscopy. we synthesize the ORF of Gal1 + BCP + T1R2 + CYC1t gene for them.

Gal1 is an Inducible promoter which is induced by Galactose. and BCP is blue cytochrome protein, we can observe it by fluorescence confocal microscopy. T1R2 is a membrane proteins which can felt sweet. CYC1t is a terminator In yeast.

From these experiments, we help BIT-China to build the part to confirm that their membrane proteins can express. here is the link to their wiki.

BIT-China helped us reconfirm whether our system can work to further validate our design. They used plates with different kinds of inducer, and detected the growth condition of bacteria with TA system.

Figure：Using plates with different kinds of inducer, we detected the growth conditions of bacteria with TA system. (A) No inducer was added into the medium, after 16 hours of streak cultivation, the bacteria grew normally. (B) 0.1mM of IPTG was added to induce the expression of anti-toxin, after 16 hours of streak cultivation, the bacteria grew normally. (C) 0.1% of arabinose was added to induce the expression of toxin, after 16 hours of streak cultivation, the bacteria couldn't grow in the medium. (D) 0.1mM of IPTG and 0.1% of arabinose was added to induce the expression of toxin and anti-toxin, respectively. After 16 hours of streak cultivation, the bacteria grew normally.

The results show that, toxin can suppress the growth of bacteria. And the expression of anti-toxin can relieve the suppression of toxin in bacteria.

This year, NKU-China constructed a plasmid contained fimS. In E. coli, the expression of the fimbriae component, FimA, is controlled in a binary fashion through the inversion of a 314 bp segment of DNA (fimS) that contains the fimA promoter. The inversion of fimS is performed by the DNA recombinase FimE, which binds to two inverted repeat sequences (Inverted Repeat Left and Right, IRL and IRR, respectively) that flank the fimS element. FimE has different binding affinities for IRL and IRR depending on the orientation of fimS, and as a result FimE is able to efficiently cause recombination only when the promoter faces IRR. Therefore, switch inversion is permanent and heritable.

Based on E.coli fimbriae control system (E.coli fimbriae (Fim) phase variation system), NKU-China have finished the switch design, to whose both ends GFP and RFP have been linked. This modified switch has been transformed into BL21 and we help them to prove it is feasible.

We added 150μl bacteria into two conical flask containing 150ml of LB medium(30μg/L kan+).And we incubated cultures in a shaker until the value of OD600 was 0.3. Then we added 150μl IPTG(100mM) into one of the conical flasks for induction. After incubating overnight, the value of OD600, red fluorescence and green fluorescence of the two strains of bacteria were measured by a microplate reader.

The results are as follows:

The results show that, when we didn’t add IPTG, the bacteria expressed more GFP. After we added IPTG, the bacteria expressed more RFP, so the fimS switch can work.

As for collaboration with TJU-China, we provided them some vital materials, and they helped us finish the indigo synthesis experiment.

This year, TJU-China utilized a novel infrared fluorescent system to track intestinal bacteria in real time. To verify their system can work in many different kinds of cells. We sent the Bifidobacterium longum and plasmid to them.

TJU-China also helped us a lot. When we used monooxygenase TfdB-JLU to degrade 2,4-dichlorophenol hydroxylase. We found that there is some blue matters when culturing bacteria. So we read a lot of literatures and datum on the internet, finding the TfdB-JLU can catalyze benzprole or tryptophan to synthesize indigo. So TJU-China helped us to verify whether our guess was right. They cultured our bacteria in LB fluid medium. When its OD600 values reached 0.4, they divided the culture into seven test-tubes. Next, they added 0.2mM IPTG in each test-tube. Then, they added different concentrations of Trp solution as follows, and cultured for 16 hours and the results are as follows.

The result shows that, in the experiment, with the increase of Trp concentration, the indigo production will increase.

This year, we collaborated with NEU-China. They did a survey about people’s knowledge of cancer and their attitude towards early diagnosis. They want to extend sample size and prevent regional biases, so we helped them hand out questionnaires in Jilin province.

The results as follow:

As the result shows, there are lots of people think detecting tumor cell depend on their VOC is not an accurate way, but there are still 34% people think the accuracy can be more than 60%. Most of people think the cancer probably can be cured if it can be detected early. Only a few people think early detection has no beneficial to the cancer.

When we asked them what their attitude towards the iSmeller are. The majority of people thought it is really convenient to detect cancer at home, they hoped this technology can be developed quickly. But a few people still suspected its accuracy. And many people hoped it won’t spend too much money. All in all, people really wanted iSmeller can work and develop a new way to detect cancer.