Team:ZJUT China B/Partnership
Partnership
Exchange and cooperation with QHFZ
Figure 1: First get acquainted with the QHFZ First of all, we both gave a brief introduction to each other 's project. The project of Tsinghua Secondary School is to use Tardigrade intrinsically disordered proteins (TDPs) to help bacteria or proteins to be better preserved in lyophilized state, and TDPs also have a protective effect on protein activity. The final product of our project involves lyophilization of proteins and the addition of TDPs reduces the effect of lyophilization on protein activity. Figure 2: Integrated exchange with the QHFZ At the same time, at the meeting, we learned about IPTG induced cell expression together, and discussed why glucose was used when it was not a good lyophilized protective agent and how to avoid using the real 2019-nCoV to validate the feasibility of the project in our project design. Based on the literature review of the TDPs by the QHFZ, they suggested that we should use SAHS33020 as an addition to lyophilized protein, and we also plan to use it to verify the protective activity of Cas protein. Eventually we decided to work together on the rest of the project, and we reached a long-term relationship.
The experimental cooperation of ZJUT-QHFZ: Figure 3: Items mailed from the QHFZ 2. We were in trouble because Cas13a seems to have a poor solubility. We used the SUMO tag to solve this problem. But the effect did not seem to be enough. We contacted a lecturer in the QHFZ, who suggested that we could theoretically improve it after iGEM based on his own experience. The TRX tag might be better than the SUMO tag, and the research paper shows that the TRX tag can improve the solubility of exogenous proteins.（Below are the literatures provided.）
ZJUT-QHFZ events：
- We met the QHFZ team at the CCIC conference in August, and started setting up meetings.
- We communicated frequently through WeChat over several months to discuss both teams’ projects.
- We helped each other with deliveries like safety forms and the promotion video
- We helped the QHFZ team on aspect of the safety form
- The QHFZ team helped us submit our promotion video when we had account issues
- The QHFZ team sent us a part that we needed to express TDPS, a protein that can protect a protease from extreme conditions
- The QHFZ team gave us advice on solubility tags.
- We helped the QHFZ team with lyophilisation experiments - there was an issue with QHFZ’s instruments. We had these instruments and helped them by using our lyophilization instruments to run experiments for the QHFZ team.
- We discussed the understanding of medal standards and poster production together with the QHFZ team.
Details:
In the project preparation stage, we hoped to get cooperation with QHFZ. Finally on August 22nd Conference of China iGEMer Community, we met with the QHFZ. After a brief understanding of each other's project, we found that our two projects have a good compatibility. So on August 30, 2020, we had an online meet up, hoping to help each other in the next period of time to accelerate each other 's project.
The help from QHFZ to ZJUT:
1. The parts of CAHS 94205, CAHS 106094, CAHS 107838 and SAHS 33020 TDPs were presented to enable us to express the TDPs, which verified our conjecture of using lyophilization and adding the TDPs for protein preservation, so that our device could be better applied in practice.
[1] Ren, R., Deng, L., Xue, Y., Suzuki, K., Zhang, W., Yu, Y., Wu, J., Sun, L., Gong, X., Luan, H., et al. (2017). Visualization of aging-associated chromatin alterations with an engineered TALE system. Cell Res 27, 483-504.
The help from ZJUT to QHFZ:
1. We helped the QHFZ to verify the bacterial lyophilization effect of CAHS 106094, SAHS 33020-CAHS 106094 and SAHS 33020 TDPs part, and solved the problem that the positive test results of QHFZ were sometimes absent due to the poor effect of the lyophilizer.
(experimental results)
2. When we were checking the experiment scheme of QHFZ, we found that they tried to use the success of the GFP expression of parallel experiments to show the success of the group protein expression, but according to the experience of the team last year, iGEM judge said "indirect experimental results can show certain feasibility, but the direct experiments are very important". Therefore, the expression of GFP cannot 100% prove the expression of the target protein, so SDS-PAGE experiment is necessary. We suggested that they use SDS-Page to directly demonstrate the expression of TDPs.
3. Due to the difficulty of protein expression and purification experiments for high school teams, QHFZ is unable to verify the lyophilization protection effect of TDPs. We plan to express and purify the protein of part 33020 donated by the QHFZ, and then carry out lyophilization experiment on the obtained protein and compare it with the lyophilized protein without TDPs, so as to finally verify the idea of using lyophilized and TDPs to preserve Cas13 protein and meanwhile help QHFZ fill in the blank of experimental data of the effects of TDPs on protein lyophilization protection. However, due to the time limitation we don't have direct results yet.
ZJUT-QHFZ in Wiki aspect:
Before the Wiki freeze, we discussed some issues together. We discussed whether their protein pages met the final criteria for gold medals. We offered some suggestions for optimizing their Wiki page.
We believe that the step-by-step argument around "why the choice was made" will be easier to convince readers and meet the medal criteria, rather than only showing the teacher's final suggestion and choice. The former can reflect more details about the choice.
ZJUT-QHFZ Mutual assistance in other aspects:
1. Before the completion of the safety form, we checked with each other to make sure there is no mistake in the filling and no omission of information, so as to ensure the accurate filling of the safety form. We also specially discussed a new problem: hazardous chemicals.
2. In the production of promotion video, we were surprised to find that the format we chose was the same, stop-motion animation. Therefore, we discussed the shooting and production skills of stop-motion animation to help each other complete the production of stop-motion animation better. When the promotion video was due, they helped us upload it to the designated submission channel, and we also answered the questions about
subtitles from QHFZ.
At the same time, we also sorted out a few points:
About stop-motion animation production:
1. The background should be fixed as much as possible. By using software, it is more convenient to locate the subsequent material and control the tiny movement of the material.
2. During the shooting process, keeping the various parameters as certain as possible can reduce the difficulty of post-editing.
3. It is important to control the number of frames in advance and shoot as many as possible to avoid the situation of frame filling.
4. If the material is very small, it will be better to use tweezers to pick it up.
For subtitles:
1. When shooting, pay attention to the background color of the corresponding position, set aside a fixed space to add subtitles, and select white words and black edges.
2. Subtitles can be switched with audio and screen to achieve the purpose of highlighting.
3. When we edited the part page of other teams, we encountered the failure of page editing. Zhang Xing of QHFZ patiently answered us and finally helped us solve this problem. This enabled us to meet the part condition of the Bronze Award and add new information to the existing part page.
4. Before the wiki deadline, we exchanged our understanding of the award criteria and we plan to discuss the poster requirements after submitting the wiki.
5. Finally, we helped the QHFZ solve the format problem of subtitle uploading.
Conclusion:
This season, the two teams have been very closely linked. Our projects are also complementary. Of course, in the future we all want to maintain this relationship and continue to study and discuss synthetic biology. In the process of our cooperation with QHFZ, we have formed a profound friendship. We not only helped each other in experiments, but also communicated with each other in other aspects, so as to overcome this special and difficult year together.
Cooperation with ShanghaiTech_China
We also cooperated with ShanghaiTech_China. We learned that their project this year was to design a device to detect antibiotic residues and antibiotic genes for doctors to diagnose. We had a lot in common in terms of project design ideas, so we both realized that further communication would bring us more progress. Therefore, we had many exchanges in the process of carrying out the project and put forward effective suggestions to each other.
Based on the use and understanding of Cas13 in our project, we believe that Cas13 can not only recognize double-stranded DNA, but also be more efficient than Cas12 in identifying single-stranded DNA and RNA, which will have great application potential in their project. Therefore, if they add Cas13 into the design, they can expand the scope of product testing, and they also fully agreed with our proposal. In addition, we provided them with plasmids that express Cas13. However, due to time constraints, they did not apply this idea to their equipment, but our suggestion did help them expand the project's potential.
In one exchange, they learned that our Rosetta (DE3) Gami used to express LwaCas13 was always contaminated with phages. Fortunately, they happened to have the competent cells of Rosetta (DE3) Gami, so they helped us do the transformation and the transformed bacteria was sent to us, which helped us solve the problem that had been troubling us for a long time, and we are very grateful for their help.
|
