Collaborating with other teams is an important part of iGEM and we try to join as many collaborations as we could. We conduct meetup with friendly teams from time to time to communicate progress. We hope that through the collaboration between the teams, the spark of ideas will be collided and a win-win situation will be achieved in the competition. It is a bit of sadness that the COVID-19 pandemic has interrupted a face-to-face collision of ideas between us, but thankfully, online brainstorming has brought us more possibilities...

In 2022, we achieved a partnership with SZPT-CHINA to promote each other in project conception, experimentation, modeling, human practice and other aspects. Throughout the competition, we maintained sincere communications and exchanges with each other (For more details: partnership).

BNUZH-China has achieved a partnership with BNU-China since last year, so we still keep close contact with each other this year. We had meetup on June 29, 2022. In this meetup, we discussed the project design and some experimental problems.

Fig.1 The online communication with BNU-China

The two teams put forward constructive opinions on each other's project, making the project design more rigorous and scientific.

Initially, BNU-China planned to put three enzymes in three different plasmids and were transformed together into the bacteria. In the case, it is hard to confirm three different plasmids are all transformed into the bacteria and the amount of enzyme expression is difficult to determine. So, we suggested that they can put the three enzymes in one kind of bacteria’s genome in order to easily regulate their expression. And we have also provided them a possible way that they can refer to: the method of transposition. Furthermore, by using different transcription initiation elements, they may control the percentage of each enzyme.

At first, we intended to use E.coli as an engineering chassis to secrete cellulase to degrade Bacterial Cellulose(BC). BNU-China proposed that the safety of E.coli as chassis could not be guaranteed, and a safer chassis could be used. Therefore, we further reviewed the literature and chose to employ the safety chassis fibroblast ATCC CRL-2522 (BJ). The engineered fibroblasts with implanted BC could not only exert photocontrolled degradation, but also promote wound repair .

We learned that BNU-China planned to use MazF as suicide switch this year. We used it as suicide switch last years and had made further characterization. We provided them the plasmid and many experiments experiences as well.

In the 2022 iGEM MeetUp in June, we had in-depth communication with the team Peking.

Fig.2 BNUZH-China and Peking participated in the 2022 iGEM MeetUp in June

After we showed our plasmid design, the Peking team said that promoter CMV has a strong ability to promote gene expression. If we put our red light-induced target gene (MazF) downstream the REDMAP system and the CMV promoter, it can easily lead to constitutive expression of target gene. So, we change the gene’s place in the plasmid. CMV promoter promote REDMAP system specifically and the MazF gene is placed upstream the CMV promoter (For more details: Engineering). Meanwhile, in order to detect and purify the red-light pathway elements and MazF expressed by cells, a purification label was added to the protein.

Fig.3 Our iterations of the REDMAP system plasmids before(a) and after(b) communication with Peking

When discussing the design of the model, we gave team Peking some suggestions that the model of their project did not only limit to the modeling of the project itself, but also to predict the behavior of the project in the co-culture system of other kinds of bacteria and yeast, so as to demonstrate the popularization of the project.

We have developed a deep friendship with SZU_China since last year, so we cooperate with them this year right along. The two teams had in-depth exchanges in the 6th Southern China Regional Meeting, the 9th CCiC and the interteam exchange meeting.

Fig.4 BNUZH-China and SZU_China participated in the 6th Southern China Regional Meeting a. BNUZH-China Project Introduction in the 6th Southern China Regional Meeting Conference Manual. b. BNUZH-China introduced the project to the teams in the 6th Southern China Regional Meeting.

Fig.5 BNUZH-China and SZU_China participated in the 9th CCiC

Fig.6 Project communication between the BNUZH-China and SZU_China

At the beginning of the project design, SZU_China attempted to produce small siRNA to achieve RNAi. When communicating with us, we pointed out that the interfere effect and absorption efficiency of siRNA by pathogens might not be high enough. We suggested that they can take certain measures to ensure the stability of siRNA, especially in the complex environment of the field. In the case, the application of shRNA may be a good idea. SZU_China decided to use shRNA as their second-generation RNAi effector molecule after integrating our opinions.

Our initial co-culture experiments of cells and bacterial cellulose membranes were unsuccessful. During the communication, SZU_China indicated that the BC film looked too thick, the alkaline substances and impurities in the membrane pores were difficult to clean, and the film was probably not washed to completely neutral on the use. As a result, the cell cannot adhere to the film and grow. After autoclaving, the membrane was not purified before co-culture with cells, and it was soaked in culture medium directly after cutting, which may still be contaminated. So, we later improved the experimental method by using a 1-2mm sterilized neutral membrane. And before co-culture, the membrane was soaked in 75% alcohol for 24h, then washed three times with PBS, and then soaked in medium for 24h. It is verified that the cells normally adhere to the film and grow well by co-culture experiment (For more details: Results).

The Bioplus-China team is committed to developing a recombinant spider silk gels as bio-ink for 3D printing of artificial skin. This system has a similar application to our project, so we have had a pleasant project exchange with their team, giving them a lot of practical guidance and help.

Fig.7 Project communication between the BNUZH-China and Bioplus-China