Team:QHFZ-China/Partnership
Partnership
Content
Jilin_China
Jilin_China and QHFZ-China helped each other a lot across this iGEM season. A gif picture below summarized our partnership.
How did we know each other?
During last iGEM season, the two teams had an unforgettable communication at the iGEM Jamboree 2019. Besides, the two teams lived in the same hotel, Hilton Hotel, and returned to China by the same flight. Therefore, the core member of Jilin_China 2019, Chen Xu Tan, and the advisor of QHFZ-China, Xing Zhang, exchanged contact information and expressed cooperation intention. Then in 2020, Tan became the team leader of Jilin_China and Zhang became the instructor of QHFZ-China, so they contacted just after the teams established.
Jilin_China and QHFZ-China knew each other in Boston during iGEM Jomboree 2019.
This year, the two teams used mostly QQ and WeChat, which are two frequently-used chatting softwares in China, to communicate. At the same time, they also used e-mail to contact during the significant cooperations for record. The emails are shown below:
1st. First communication
In May, the two teams met for the first time online. The meeting aimed at being familiar with each other. The epidemic seriously affected the processes of both teams. So they had a talk about how to work during the epidemic. As the two teams come from different regions of China, they reached a consensus that if one team had some problem to enter the laboratory months later, the other would offer some help in wet lab. Besides, they both had some candidate project designs during that time. For example, QHFZ-China had four: bacteria storage, HBV detection, bacteria battery and food spoilage detection. We exchanged ideas to help each other choose the topic. As far as we known, that the great interest of us to the ‘life game’ project influenced the topic selection of Jilin_China.
2nd. Questionnaire distribution
In June, Jilin_China had a trend to choose the ‘life game’ topic. They carried out a questionnaire to investigate the reaction of the public to the ‘life game’. As we are high school students, it is indicated that if we distributed the questionnaire, more answers from high school students would be collected, which Jilin_China lacked. Therefore, the age composition of the answers would be more reasonable. As a result, Jilin_China communicated with us and we helped them distribute the questionnaire via our blog. Jilin_China told us that the result was ideal.
We edited a article in our blog (left) to distribute Jilin_China's questionnaire (right).
3rd. Interview with a structural biology expert
In June, QHFZ-China found the structures of TDPs should be studied to guide their experimental design. However, as high school students, it is too hard for us to study the protein structure. Fortunately, Guangyuan Song, who was the team leader of Jilin_China 2014, now is a Ph.D candidate of structural biology. Jilin_China helped us contact Guangyuan Song, and then we communicated with Song for several times across the summer vacation. Indeed, later, in August, when we wanted to combine different TDPs, we utilized structure prediction to choose the target TDPs. It is clear that without the help of Jilin_China and Guangyuan Song, we could not optimize our design.
Left: We invited Song from Jilin_China 2014 and we wrote a record in our blog. Right: He helped us choose the target proteins via structural biology.
4th. Meetup
In July, Jilin_China held a meetup, Northeastern coastal areas online Meetup . We really appreciated that they invited us to join it. Among the teams, we were the only high school one. During that, we learnt a lot from the other teams’ presentation and our team members, Xinyuan Wang, Yeqi Wang, etc. asked a lot of questions. when we asked questions, they gave answers with enthusiasm. Sizhe Duan introduced our project to the all participants and they gave us a lot of advice. Via the meetup, we met many teams and had collaborations later, such as NEFU-China and BUCT-China. In addition, we wanted to hold a meetup for high school teams. However, we did not have enough experience and did not know how to hold a meetup well. Jilin_China taught us how to do it well by the meetup and gave us many tips. Then on Aug. 1st, we successfully held our meetup, ‘Beijing International iGEMer Collaboration Seminar’.
Top-left: The meetup held by Jilin_China and we attended it. Top-right: Our presentation PPT. Bottom-left: Our script. Bottom-right: The meetup we held with the guidance of Jilin_China.
5th. A combination of the two projects in theory
After the meetups above, one of our members, Yeqi Wang, came up with an idea. Computer games can be paused and archive. However, usually the lives cannot be paused. The project of QHFZ-China is to achieve a new storage method for bacteria, using TDPs and freeze-drying to pause the metabolism of bacteria (for more information, please see the Description page). Can we help Jilin_China archive their ‘life game’? We had a short talk with Jilin_China, we were very excited. For them, saving the game is a big problem and we solved it; for us, our project had one more application scenario. However, because we had not so much data to support each other’s own project at that time, we decided to talk about the idea months later.
6th. Sharing and measuring the arabinose sensor
In August, as our work of wet lab progressed, we met a problem. We needed to construct a gene circuit to degrade TDPs before using the engineered bacteria, so that TDPs could not affect the functions of the bacteria. As we use lac operon to regulate TDPs, we should use another operon to regulate the protease, mf-Lon. As a result, we need to construct such a sensor and prove that it can work. Jilin_China happened to use the Arabinose operon this year. They kindly promised that they were willing to share it with us. What’ more, their advisor, Yu Ma, gave us a lesson about the arabinose sensor. After we got the part, we measured it. The data helped us proofed the feasibility of our concept. Meanwhile, Jilin_China only measured the part at three arabinose concentrations. We helped Jilin_China measured the part at more arabinose concentrations. Our measurement also means that the part works well in another lab and with another protocol. All these helped Jilin_China prove that their gene part is an excellent one for widely use. Both teams benefited a lot during the cooperation.
Top: The plasmid they sent to us. Bottom: The sequences of the two core regions in the plasmid.
Left-Top: The gene circuit of our final aim, which showed that we need to test an arabinose sensor. Left-bottom: The data that we measured by the part Jilin_China gave us. Right: Some data points were strange, which we were not quite sure. If we delete these points, we will get this picture. We put the both pictures here, aimming at avoiding misguiding other iGEMers.
7th. Modeling
In September, we realized we would have no enough time to finish our TDP degradation system. We decided to construct a math model to simulate it. However, after we listed the formulas, none of us could use matlab software to solve it. Jilin_China helped us wrote two m files with ‘ode’ function. Then we adjusted the parameters and got the final result.
Top: Two differential equations in our model. Bottom-left: The m files that Jilin_China helped us write. Bottom-right: The computational results of the m files before we adjusted the parameters.
8th. Subtitle
In September, when the deadline of promotion video was coming, QHFZ-China met a problem. The system told us that our subtitle was invalid. At that short time, we cannot find why. Jilin_China gave us some advice and we corrected it in time. Later, they also suggested to use AU to make the subtitle of the final video.
Our subtitle of the promotion video was invalid at first. Several teams, of course including Jilin_China, gave us many advice and saved us.
9th. Proof of QHFZ-China’s concept
In October, QHFZ-China got into trouble. On Sep. 26th, QHFZ-China helped team NEFU-China 2020 make bacteria dry powder and then we sent the powder back to NEFU-China at room temperature. The aim is to prove that our method is useful and convenient. However, during the following half month, NEFU-China failed to recover the bacteria, which means that the bacteria could have died before being recovered. This is an extremely bad news to us. NEFU-China told us that they began to doubt whether our freeze-drying method is resultful. At the same time, we were worried that our design is useless, which means we would fail in this iGEM season. However, as it was only 20 days away from the wiki freeze day, we were afraid we had not enough time to find where is wrong. As a result, we sent some other bacteria powder we made to Jilin_China. Jilin_China helped us do the experiment. Fortunately, the bacteria were successfully recovered though the powder was made about 1 month ago and room temperature transportation was used. In conclusion, Jilin_China confirmed our project in their lab other than QHFZ-China’s lab. It's worth mentioning that the help occurred at a key moment of QHFZ-China.
Left: The bacteria powder we were ready to send. It wrote that the powder was made on Sep. 13th, nearly a month before the recovery test. Right: The powder was received by Jilin_China.
Top: The powder was recovered a month after freeze-dried. Bottom: Jilin_China helped us proved that the bacteria powder could be revived and CAHS 106094 (a TDP) helped enhanced the survival rate.
10th. Final communication
Just several days before wiki freeze, the two teams had a final meeting. They talked about wiki, presentation, poster, etc. This year, several new pages and criterions came out, and we do not have enough confidence fully understanding them. To avoid mistakes, we shared our opinions about them. For example, we talked a lot about the page contribution and engineering success. We also came to an agreement that if we had some problem for the next several days, we will try our best to help the other teams. Of course, we reviewed what we collaborated this year, to wrote the partnership page without missing points.
Previously, we told each other that we would talk about the combination of our projects in the future. This
time we designed it together. Thanks to the summary from Jilin_China, our imagination is as following:
If players need to pause the ‘life game’ (Jilin_China’s project) while playing the game, they can use the
freeze-drying storage method of QHFZ-China to pause the whole system, thus achieving the effect of "game
saving". Because Jilin_China’s system consists of nutrients, autoinducer for quorum sensing, bacteria itself
and other components, using the traditional centrifugal supernatant and restoration method obviously
destroyed the whole system. Through direct freeze-drying method designed by QHFZ-China will avoid the
negative impact on the system components. To continue the game, you just need to add certain amount of ddH2O
to recover the system. Although such an idea is still a long way off, it is theoretically possible for
Jilin_China to archive the game and shows the potential value of the project of QHFZ-China.
Summary
This year, Jilin_China and QHFZ-China had an amazing cooperation. As a high school team, there is a distance between us and undergrad teams. As a result, Jilin_China gave us more help than that we gave to them. However, we tried our best to offer help. The two teams planned to collaborate since iGEM Jamboree 2019. When the new criterion came out, we realized that our cooperation should be a partnership, which is more than collaboration. Therefore, we went on our cooperation. We connected more and more. Our cooperation included wet lab, dry lab, wiki, human practices, videos and many other fields. The cooperation truly helped both teams a lot. We hope the cooperation model will continue in the next year and bring inspiration to other teams.
ZJUT_China_B
ZJUT_China_B and QHFZ-China helped each other a lot across this iGEM season. In 2017, the grand prize winner of iGEM overgrad group, Team TUDelft, studied Cas13a protein and TDPs. We got the inspiration from TUDelft. Though we mainly used TDPs to protect live cells this year, TDPs could also be extracted and protect purfied proteins. Luckily, we met ZJUT_China_B, which team used Cas13a to detect COVID-19 this year. Threfore, We paid high attention to each other and began the partnership.
Getting acquainted with each other
ZJUT_China_B and the QHFZ-China attended The 7th Conference of China iGEMer Meetup in August and found that the two teams were very suitable to collaborate. To ZJUT_China_B, they needed to freeze-dry the Cas13a protein to prolong the shelf time of their product, so they needed to use TDPs to keep the activity of protein activity. To QHFZ-China, we expected to test the freeze-drying method to protect bacteria in another laboratory to verify our result. However, it was very hard to find a team that used lyophilizer (freeze-drier) equipment. Therefore, to collaborate with ZJUT_China_B is a rare opportunity to prove our design. The two teams decide to continue communication after the 7th Conference of China iGEMer Meetup.
First one-to-one meetup
On Aug. 30th, we had an online one-to-one meetup. We respectively gave a talk to introduce our project designs. Secondly, we gave each other some advice. For example, ZJUT_China_B suggested us that we should improved our experiment design. We should prolong the storage time at room temperature before testing the survival rate. We suggested that COVID-19 is very dangerous, so ZJUT_China_B should pay a lot of attention to safety, though they did not use live virus. Thirdly, we discussed how to collaborate. During that time, our results were not quite stable. We thought the reasons could include unstable lyophilizer or our unskilled experimental technique. Thus We hoped ZJUT_China_B could help us reappear our result to prove our concept. ZJUT_China_B promised to try it. ZJUT_China_B wanted to use TDPs to protect their Cas13a protein during freeze-drying. Indeed, during design the gene parts, we had a talk with our instructors and advisors, they advised us to use T7 promoter and add a 6xHis tag into TDPs. As a result, luckily, they could use the vectors we constructed to express by E. coli BL21 (DE3) strain and purify TDP by Ni-chelating affinity chromatography. Then, we studied five TDPs this year. However, as time limited, we discussed which one to use. With the help of our literature research, we adivsed that SAHS 33020 should be chosen, CAHS 106064 was also a candidate. We expected our partnership a lot.
Wet Lab
We prepared the plasmids as fast as posible and sent them to ZJUT_China_B. ZJUT_China_B received it and started the experiment at once. We shared the protocols and they tried several times. However, none of the experiment was easy, such as freeze-drying and protein purification. Fortunately, at the end, some data came out and benefited both teams.
The plasmids we sent to them.
The helps from ZJUT to QHFZ
(1)They helped us verifying 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 our team were sometimes absent due to the poor effect of the lyophilizer.
(2) When they were checking the experiment scheme of us, they found that we 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. They suggested that we should 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, we were unable to verify the lyophilization protection effect of TDPs. They plan to express and purify the protein of SAHS 33020 donated by the us, 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 us fill in the blank of experimental data of the effects of TDPs on protein lyophilization protection. However, due to the limitations of time, they don't have direct results yet.
Daily connection & Project design
We have some ways to communicate, such as WeChat and Feishu softwares. We talked a lot across the season. We talked about human practices, art, wiki, etc. However, of course, we talked project design a lot. Here we listed some tips.
（1）ZJUT_China_B adivsed us to prolong the storage time at room temperature before testing the survival rate, which could better simulate the practical use. As a result, we tried to store the bacteria powder for 10+ days and 1 month. The results better proofed our concept.
（2）ZJUT_China_B found that glucose is not a good lyoprotectant and asked why we used it in our project. We explained that it is beneficial for us to observe the effect of TDPs and relatively cheap. In the future, when TDPs are used in real world, better lyoprotectant will be used to cooperate with TDPs.
（3）ZJUT_China_B got in trouble because the solubility of Cas13a protein seemed bad. They use a SUMO tag to solve it. However, the effect seemed not enough. To give a better solution, we connected our instructor and let him offer help by his experience. In the end, we gave them a tip: TRX tag may be better than SUMO tag [1]. The advice improved their design in theory.
The research paper that shows TRX tag can improve the solubility of exogenous proteins.
Safety form
Before the deadline of safety form, we checked and discussed each other's. We made sure that our safety form were integrated and clear. We specially talked about a new question: about the hazardous chemicals. We cannot surely distinguish carcinogens and mutagens. Thus we discussed and searched information together. At last, we finished and iGEM foundation gave us an e-mail, telling us that our safety form is OK.
Promotion video and subtitle
Both teams planned to use stop motion animation to make the video, so the two teams communicated some details.
Before the deadline of promotion video. The subtitle of QHFZ-China was judged invalid by the system. We were quite nervous and asked many teams for help, of course, including ZJUT_China_B. ZJUT_China_B gave us a lot of advice and finally we corrected the subtitle.
During that time, ZJUT_China_B was busy at moving from one campus to another, having no time to finished the video in time. QHFZ-China helped them contact iGEM video group to explan the condition. Days later, ZJUT_China_B finished the video, we checked their video and gave advice to optimize their audio. Then ZJUT_China_B found they cannot upload it. We helped them upload it by our account.
Wiki
Before wiki freeze, we talked about some related issues. We asked if our protein page meets the last criterion of gold medal. They gave some advice to optimize that wiki page.
Summary
This season, the two teams connected very close. Our projects were very suitable to collaborate. Obviously, we want to keep the relationship regardless of the end of this season. As we are high school students, we lack much knowledge. Undergrad team ZJUT_China_B gave us a lot of support. We really appreciate them and expect the partnership next year.
References
[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.
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