Team:iBowu-China/Partnership
Partnership
Partnership with CKWA-China
CKWA-China is new to iGEM in year 2021. After their team established, CKWA-China's art designer Jason learned that his friend Angela Tang was the art designer of team iBowu-China. Our teams quickly got into touch and helped each other throughout the iGEM season on experiments, Human Practices, outreach, promo video and so much more! Check out the timeline below that summarizes our partnership!
A Brief Introduction of Team CKWA-China
All members in Team CKWA-China are students from an international curriculum school in Beijing, namely the Chaoyang KaiWen Academy. The team members are from grade 6 to grade 10, and this is the first time ever they joined iGEM! The team leaders learned about molecular biology in their school club and they discovered iGEM when they pursued their interests in this field. For this year's project, the team had their eyes on treating cancer recurrence with a special protein.
April
Meetup: Team iBowu-China and CKWA-China's first contact
In April, Team iBowu-China initiated the first meetup in China, to exchange ideas of team establishment and project brainstorm. With the help of CCiC (), the meetup event was advertised over their official WeChat channel and received warm receptions quickly from many teams, including high school teams and college teams. Team CKWA-China was eager to learn about iGEM and thus applied to enter the meetup through our WeChat channel, and they worried about the presentation since they didn't have past projects nor project ideas. We as the host team were so glad to expand the iGEM community, and registered them into our official WeChat contact group (Figure 1.b), and let Team CKWA participate without a presentation but also encouraged them to communicate actively on the meetup event.
Figure 1: CKWA-China members attended the 4/18 Beijing Local Area Meetup.
On the meetup, iBowu-China and CKWA-China talked about details of project design, experiments, and human practices. We suggested CKWA-China team should work on E. coli because of their short life cycles. CKWA-China also showed their creativity in project ideas and human practices activities. Therefore the two teams decided to be partners throughout the iGEM 2021, and scheduled a follow-up meeting to move forward with the project ideations.
Follow-up: iBowu-China's 2020 experience and CKWA-China's project
We scheduled an online meetup between only our two teams on Tecent Conference (or Voov) to advance both teams' project brainstorm. We introduced all of iBowu-China's project ideas this year, including but not limited to mitigating cockroach disasters, developing depression cures, synthesizing effective ingredients of herbal medicines and so on. Since a few of our members also competed in 2020 iGEM, they also introduced the iBowu-China 2020 project Sureloc which aims to treat cancer cells with targeted drugs.
New to the field of synthetic biology, CKWA-China exhibited strong interests in treating cancers. After they learned that iBowu-China only received a silver medal for the 2020 project in developing cancer drugs, CKWA-China decided to attack the big problem from a new perspective: the recurrence. Our members also educated CKWA-China members on how to find relevant information from the vast online sources and showed them how to do literature searches.
May
Regular Contacts: moving forward altogether
iBowu-China tauch them iGEM is more than experiments, but also about HP and education and safety. During this time, we held online meetins regularly. The below picture from one of the meetings show when iBowu-China talked about the value of Human Practices to CKWA-China members. The second person in the first row is student leader Rachel from iBowu-China, the first person in the second row is the associate student leader Haber from iBowu-China. The other attendees are CKWA-China members.
Lon-protease: CKWA-China project decided!
CKWA-China team found a fresh research paper Lon-protein [1], and they were excited to invite us over to discuss the project. iBowu-China help CKWA-China evaluate the feasibility of the project. Our team members Lucas and Linda, experiment group leaders from the past year, thought highly of the project idea. The project can be done if CKWA-China can successfully produce the protein in E. coli. CKWA-China found out Lon protein can be synthesized in E. coli from Abcam, a molecular biology company that manufactures biological materials.
Harry is the student leader of CKWA-China. He stood at the board and introduced the Lon protease paper to both teams. Team iBowu-China members, Lucas Ji, Joyce Jiang, Haber Chen, Rachel Chen listened carefully to their presentation in the back.
This is the Lon protease paper. References are given below the wiki page.
Engineering: iBowu-China helped CKWA-China with details
iBowu-China suggested that CKWA-China try pet28a+ plasmid with BL21(DE3) strains, which was the designed expression system for iBowu-China's 2021 project. iBowu-China team experiment leaders help CKWA-China build their plasmid vector based on our own bG-pet28a plamid. We also proposed that if the expression of Lon protease were successful, CKWA-China could consider the use of the liposome for delievery, which was fully studied by iBowu-China team in 2020 project.
Meanwhile, CKWA-China offered the help to test and measure the performance of T7 promoter used by iBowu-China this year. iBowu-China team had some success by this time using T7 promoter for the expression of the beta-glucuronidase enzyme, and then was planning to adjust the strength of this promoter by mutating some of the promoter sequences. If the new promoters were successful, CKWA-China could use the mutated promoters to upgrade their yield of Lon protein, and at the same time, CKWA-China will provide more data through measurement of new promoters on the expression of Lon protein.
Figure 4: Plasmid Lon-protease of CKWA-China.
June
Safety-Form: always needed a second opinion
It is deadline time! iBowu-China had previous experience in filling out safety forms, and we guided CKWA-China on the concerns of lab safety and biological safety, how to prepare for lab safety and how to correctly fill out the parts in excel sheets. CKWA-China team doubled-checked our safety form and pointed out iBowu-China had better claim the safety measures regarding mammalian cells, namely the possible use of HeLa cell in testing if our product, glycyrrhetinic acid, had the effect on suppressing cancer cell growth.
Promo Video: brainstorm for better demonstrations
Back-to-back submissions of Safety Form and Promo Video were not easy, but we were thrilled and productive. The two teams talked about promotional video ideas and exchanged information on the filming resources and techniques. iBowu-China shared a lot of background information and graphical materials regarding cancer and cancer treatment for CKWA-China's project introduction in the video. CKWA-China put us in contact with a great filmmaker studio, and put forth a few fun ideas of scenes for iBowu-China's video, including the ending of our promo video where the team formed a circle on the floor.
July
Starting the Lab: iBowu-China tutored and CKWA-China delivered
We were happy to train the CKWA-China team on lab skills. Starting from preparing LB culture, pouring LB agar plates, to pipetting the proper amount of PCR mix and DNA templates, we tutored CKWA-China team to jump-start their lab tour. CKWA-China team members were good learners and humble practitioners! They prepared about 50 agar plates that would be much needed by us iBowu-China because we planned a lot of promoters substitution and tests of conditions. In the beginning, everyone would make naive mistakes and could use a double-check. For example, CKWA-China pointed out our mistake in the plasmid extract that an iBowu-China student forgot to add ethanol in preparing the solution; and we held regular meetings every week to talk about (actually to fret about) our slow experiment progress and check for mistakes in lab operations.
Sharing for More: CKWA-China shared a great Human Practice opportunity
Both teams wanted to learn about the use of their product for curing cancer. CKWA-China had a chance interviewing medical experts and both teams attended the meeting. We reached a conclusion that it is not feasible to make our products into delierable drugs within iGEM season.
Distribution of Survey: CKWA-China helped iBowu-China on BioDoodle survey
CKWA-China helped iBowu-China on BioDoodle survey. The survey was intended to learn about what elementary school kids and secondary school kid desire to study on the subjects biology. CKWA-China's school has both department and the team distributed the survey and helped iBowu-China collected feedbacks.
Optimizing Protein Expression:
CKWA-China had difficulties in expressing their protein in that the production yield was really low. We iBowu-China tried the experiments ourselves and could not find a solution. Our members reached out to a protein expert, CEO Dr. Wang from AIDE Pharmaceutical. Team members Linda and Rachel and (those not covered in the picture) David Shen and Lucas Ji from iBowu and Juicy and Jason from CKWA-China attended the Human Practice interviewing Dr. Wang. Dr.Wang provided production optimization ideas and asked both team members to study more about the structure details and function details of the protein we planned to use. He suggested the possibility that the Lon protein may be degrading the normal proteins and enzymes in the E. coli production strains, and suggested we should try to optimize induction conditions at different temperatures (16C, 25C or room temperature and 37C) and also different concentrations of IPTG.
Team member Linda and Rachel and (those not covered in the picture) David Shen and Lucas Ji from iBowu and Juicy and Jason from CKWA-China attended the Human Practice interviewing Dr. Wang from AIDE Pharmaceutical on the protein production.
September
Education on Campus: CKWA-China and iBowu-China education in Kaiwen Academy
CKWA-China planned an education activity on campus for the middle schools. They invited iBowu-China to talk about herbal medicine, iGEM experience and introduced the BioDoodle.
Safety Form Finale
October 1st is the final day for submitting safety forms. iBowu-China and CKWA-China checked and discussed the safety forms with each other to make sure there is no missing pieces. Both teams received a clearance from iGEM headquarter after the preliminary submission in June, but we were careful in finishing the safety forms. Especially we checked the parts we planned to submit to make sure there were no mistakes. This deadline was also for title and abstract submission, too! We proofread each other's titles and abstracts and offered ideas for revisions. iBowu-China had more experience in summarizing the research work findings and pointed out 1) that CKWA-China should add rigorous numbers to their abstract when claiming the c-Myc to be one of the most important factors for the specific cancer types, 2) that CKWA-China should write in a more objective manner in the abstract for stating facts and suggested re-wordings and 3) that CKWA-China had better put their future plan into the abstract with one or two sentences so readers could better understand where the project would lead to.
October
All-in for the Freeze: help before the wiki deadline
Teams can't be more busy than before the wiki freeze! We took our last chances to raise suggestions for each other's wiki presentation, holding an online meeting. We really wanted to do mock interviews and mock judging sessions, but we are afraid that this event has to be put off after our wiki freeze!
Partnership with NWU-CHINA-B
May
Meet-up:
This was a general meetup of Northwest China online meeting. Many college teams and high school teams presented their project ideation and asked around for potential collaborations. In the manufacturing session during morning meetups, iBowu-China presented the project idea of using synthetic biology method for producing the effective ingredients in licorice, and NWU-CHINA-B talked about their research and efforts in making ginsenoside, which is the effective ingredients in ginsen. Our teams quickly realized we were the natural partners as the two teams both focused on the herbal extract with pharmaceutical effects. Right after the presentation session, we chatted in the online meeting chatting area and established a WeChat working group, exchanged our PPT slides for better understanding each other's projects and scheduled for a follow-up meeting.
Regular Contact
During the entire iGEM season from May to October, the two teams remained close and regularly contacted each other, because both teams were targeting research in Chinese herbal medicine, targeting the improvement of herbal products, and we discovered similarities and directions for deeper collaboration between our two teams' projects. We followed up on each discussion on WeChat chatting, in Email exchange, and sometimes in online conferences. Every aspect of the iGEM project was up for discussions, like experimental process, success and failures, mathematical modeling, and the refinement and checking of safety forms. This has been a great boost and improvement to the progress of the project between our teams!
Partnership on Human Practices with NWU-CHINA-B
iBowu-China has been planning for a documentary introducing traditional Chinese medicine, and we asked NWU-CHINA-B for help. They provided us with information on ginsenosides. At the same time, BioDoodle project was fast-moving and we asked NWU-CHINA-B team to help contact some teams who were interested in participating in as much collaboration as possible. NWU-CHINA-B kindly promoted our BioDoodle project to many teams.
We mailed NWU-CHINA-B a product for education - herbal scented sachets, as a testimony to the friendship between the two teams. At the same time, we shared all of our interview notes from the HP events, of which all were related to herbal medicine, including professors and companies.
On experiments
iBowu-China asked NWU-CHINA-B about the operation of the HPLC liquid chromatograph that we were unfamiliar with, and NWU-CHINA-B helped the iBowu-China team step by step and solved this problem. NWU-CHINA-B team members taught the iBowu-China team online + on-site in the lab and also shared NWU-CHINA-B's current product recordings using HPLC with us iBowu-China team. In addition, NWU-CHINA-B shared with us their work regarding protein structure prediction, which inspired us iBowu-China team to consider deeper about where genes can bind through protein structure prediction. At the same time, NWU-CHINA-B has chosen to match their project with the Pichia pastoris and use the AOX1 promoter, which better enhances the expression.
As the concentration of plasmids coming out of electrophoresis was too low, we iBowu-China team helped NWU-CHINA-B to analyse the reasons for the failure of electrophoresis. Then we exchanged our enzymes and tried to catalyze each other's reaction substrate, such as on ginsenoside Rb1 by our iBowu-China β-glucuronidase enzymes, but it did not work on NWU-CHINA-B's substrate either.
Events Together
Together, we participated in the 2021 iEEE International Conference to educate the general public and popularise the science of synthetic biology, to introduce young people and scholars from other research fields to the wide impact of synthetic biology in the world, and to present the projects of our two teams. The response from the general public has been very positive and new to the field of synthetic biology, and the educational outreach by our two teams has resulted in a group of young people who are intensely curious about the synthetic biology community and we encourage them to continue their exploration.
Partnership with SHSBNU-China
Getting to know each other
iBowu-China met SHSBNU-China in the CCiC Beijing region meeting and had kept in touch ever since. We iBowu-China contacted SHSBNU-China in September to find a way of purifying our targeted protein knowing that SHSBNU-China team has built a fusion protein that has the potential of using its cellulose-binding domains to bind cellulose, thereby purifying the linked domain, in our case, β-glucuronidase.
Two teams met up and discuss project details.
SHSBNU_China proposed to iBowu_China of using cellulose suspension as a substitute for the ordinary Ni-NTA beads and substitute the His-tag with CBD domains on each end of the enzyme. SHSBNU_China team deleted the original Streptavidin domain from the protein and substituted it with iBowu_China’s protein. We iBowu-China sent SHSBNU_China the plasmid and SHSBNU_China added it into cellulose-binding platform and transformed into Escherichia coli. SHSBNU_China then sent it back to iBowu_China’s laboratory for further analysis.
The first trial of the induction of the protein was not successful and no protein was produced. The second trial was carried out with the induction intensity same as what SHSBNU_China used in the expression of BBa_K3798032. Due to lack of time, the purification had not experimented. However, the efficacy of the fusion protein was examined. From the result, it can be concluded that the β-glucuronidase fused into CBD platform is still viable and can perform its job of catalyzing glucuronide.
Therefore, for SHSBNU_China, this has proven that CBD platform would not distort the function of the original protein that is linked.
For iBowu-China, we found a viable way of immobilizing our enzyme
In the future, we hope to continue the partnership with iBowu_China to finish the purification experiment and make the mass production of glycyrrhetinic acid feasible.
References
1. Butler, D.S.C., Cafaro, C., Putze, J. et al. A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer. Nat Biotechnol 39, 754–764 (2021). https://doi.org/10.1038/s41587-020-00805-3