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 have a better way in testing the expression of membrane protein.
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)in the front of the coding reigon of T1R2 and it could be observed the protein T1R2 on the membrane by confocal fluorescence microscopy. We synthesized the ORF of Gal1+BCP+T1R2+CYC1t gene for them.
Gal1 is an inducible promoter which is induced by galactose. BCP is blue cytochrome protein, it can be observed by fluorescence confocal microscopy. T1R2 is a membrane proteins which can sense galactose. 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 check whether our system could work to validate our design. They used plates with different kinds of inducer and detected the growth of bacteria with TA system.
| Plate A | Plate B | Plate C | Plate D | |
|---|---|---|---|---|
| 0.1% Ara | — | — | + | + |
| 0.1mM IPTG | — | + | — | + |
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 containing fimS. In E. coli, the expression of the fimbriae component, FimA, is controlled in a binary fashion through the inversion of a 314 bp DNA segment (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, 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 has finished the switch design, to whose ends both GFP and RFP have been linked. This modified switch has been transformed into BL21 and we helped them to prove it is feasible.
We added 150μl bacteria into two conical flasks containing 150ml LB medium(30μg/L kan+).And we incubated cultures in a shaker until the value of OD600 reached 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:
| Vector | No IPTG | Add IPTG | |||||||
|---|---|---|---|---|---|---|---|---|---|
| OD600 | 0.762 | 0.723 | 0.802 | 0.746 | 0.762 | 0.778 | 0.587 | 0.604 | 0.594 |
| GFP(472/512) | 12 | 17 | 9 | 3487 | 3193 | 3376 | 151 | 122 | 132 |
| RFP(587/610) | 14 | 15 | 13 | 331 | 346 | 275 | 997 | 960 | 852 |
| GFP/OD600 | 15.75 | 23.51 | 11.22 | 4674.26 | 4190.29 | 4339.33 | 257.24 | 201.99 | 222.22 |
| RFP/OD600 | 18.37 | 20.75 | 16.21 | 443.70 | 454.07 | 353.47 | 1698.47 | 1589.40 | 1434.34 |
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.Here is the link to their wiki
NKU-China also did some experiments for us. As for our project, two significant changes will happen when geneguard takes effect. One is the delaying in bacterial growth, the other one is transformation in bacterial morphology. The two experiments we designed above certified geneguard’s function through delaying in bacterial growth. We also wanted to demonstrate it through transformation in bacterial morphology. However, we lacked appropriate equipment for observation. Thus, we seek help from our partner NKU-China.
We designed the morphology experiment according to our pervious work.
The results are as followed:
figure 2. Groups of control, 0.2% arabinose induction, 0.2% arabinose and IPTG co-induction were set. Bacterial morphology was observed in 1.5h, 3h, 6h.
As Figure 2 shows, bacterial shape in control group didn’t have evident change in 1.5h, 3h, 6h, all in rhabditiform. Arabinose induction group appeared bacilliform in 1.5h. in 3h observation, part of bacterial turned into roundness. In 6h observation, most of bacteria became rounded. The arabinose and IPTG co-induction group appeared bacilliform, in 3h observation, part of bacterial turned into roundness. In 6h observation, most of bacteria were bacilliform.
E.coli in control group didn’t express inducted heterologous protein. Thus, they appeared bacilliform. While in arabinose induction group, part of bacteria became rounded in 3h and most of them turned into roundness in 6h. we can consider that toxin CbtA led to the transformation in bacterial morphology.
Part of bacteria in Arabinose and IPTG co-induction group turned rounded in 3h observation. That’s because the toxin addicted time was too short that only part of bacteria expressed cbtA. While expression of cbeA made most bacteria appeared in bacilliform in 6h observation. Because the experiment time was limited, we didn’t get the distinct result. We think that more solid result will appear if we extend the observation duration.
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. Here is the link to their wiki
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.
| Tube No. | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
|---|---|---|---|---|---|---|---|
| CTrp(g/L) | 0.1 | 0.25 | 0.4 | 0.6 | 0.7 | 0.85 | 0 |
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 depending 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. Here is the link to their wiki
Since we wanted to get the feedback of our project, in September, after we got some results, NEU-China helped us interview associated Prof. Peiyong Liu, vice Dean of Northeastern University in China. They introduced what we did, and discussed with professor. Then, they asked her some views towards our project. She thought that the usage of Geneguard system in an innovative idea and it was feasible to control the engineered bacteria in the environment. She also thought that our project was meaningful when concerning biosafety issues. Solving this problem using synthetic biology methods was a nice attempt. She spoke highly of our project and wished the best for us. Her suggestions were meaningful to us and gave us confidence. We appreciated NEU-China for the interview.
On 12th of August, we had a voice conference with Tianjin, XMU-China, FAFU-China, SJTU-BioX-Shanghai, SCUT-China_A, and UCAS. After the conference, we decided to build an alliance and a worldwide database for the contents of heavy metals in local soil. We took charge of sample collecting in the northeast China. Because of the huge sample size, we still continued to do this work after wiki freezing. We hope we can build the database in China, and guide our future work.
Here are the other teams' wiki:
Tianjin XMU-China FAFU-China SJTU-BioX-Shanghai SCUT-China_A UCAS
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