No man is an island, entire of itself; every man is a piece of the continent, a part of the main; ——John Donn
Success is inseparable from a good communication and cooperation. We have established long-term partnership with NWU-CHINA-A, HBUT-China, BNUZH-China and NEFU_China this year, and we have been cooperating in many aspects such as HP, Experiment, Model and Wiki.
We decided to continue our partnership with NWU-CHINA-A this year based on the good relationship we have had with NWU-CHINA-A for many years.
We conducted our first online communication on July 19th. First, we introduced each other project and explored cooperation opportunities. We discussed the ideal form of our products due to our projects are presented in the form of live bacterial products. Immediately after that, they were interested in the kindergarten education activity we did, so we shared with them the specific process of that activity. Finally, we also set up a WeChat group and decided to have regular meeting.
NWU-CHINA-A invited us to participate in the 4th Northwestern China iGEM Meetup that they hold on July 23rd. At the meeting, We received useful guidance from the invited guests, such as the style of our promotion video is similar to last year's project, so explanation is required; the background introduction should be more professional; children's photos which were exhibited on wiki pages should be approved by children's guardian. NWU-CHINA-A was interested in how we could contact the kindergarten, so we shared our experience on how to contact them (by phone).
NWU-CHINA-A helped us to find Dr. Penggao Dai, the technical director of Shaanxi Lifegen CO. ,Ltd., when they learned that we are having difficulties in finding a company because of the COVID-19. We used Tencent Meating to communicate with Dr. Penggao Dai online. During the meeting, Dr. Penggao Dai was positive about the design of our project. He suggested us to use the activation solution and freeze-dried bacterial powder to solve the difficulty when we were confused about how to keep the bacteria active on Augest 10th. This inspired us about the design of the product.
We invited NWU-CHINA-A to participate in a joint five-team online science lecture "Enter the World of Synthetic Biology" that we hold on August 6th. They shared the application of synthetic biology in the treatment of cancer in the popularization lecture.
We conducted the Five-team Meetup at the end of the science lecture. We shared with them what we learned from our trip to Yakult. We found that not all probiotics can survive the acidic environment of the stomach and reach the gut to play a role, and their project is to detect gut micro bleeding through engineered bacteria, and it may also encounter the above situation. Therefore, we suggested them to simulate the gastrointestinal environment for strain screening, and we also provided them with the contact information of Yakult.
NWU-CHINA-A indicated that the experiment encountered difficulties in the regular meeting on August 5th. The bacteria ( E. coli DH5α) containing the pigment protein pellet could not be cultured. To help them we asked for our instructor Rongrong Zhang for help on August 6th. Please see below for details.
The plasmid containing the pigment protein amliCP was transferred into E. coli DH5α for amplification and incubated overnight.
Take 100μL of freshly prepared receptor cells, add 1μL of recombinant DNA ligation product, mix by blowing on ice, and place on ice for 30min.
Put the tube to 42°C for 90s of heat excitation.
Ice bath for 2min.
Add 800μL of LB liquid medium, incubate at 37°C, 150rpm for lh (slow shaking).
The cells were collected by centrifugation at 12000 rpm for 5min, and about 100 μL of medium was retained. The excess supernatant was discarded, and the cells were resuspended by aspiration and coated in a Petri dish containing chloramphenicol (100μg/mL).
Take 20, 30, 50μl of bacterial solution to be coated in solid medium, respectively, and invert the flat dish at 18:00 and incubate at 37℃, but no colony generation was observed, what could be the reason?
First, the concentration of chloramphenicol (100μg/mL) used in NWU-CHINA-A is high, the concentration of antibiotic we generally use in E. coli is (18μg/mL). Secondly, when you construct the plasmid, the promoter region in front of the chloramphenicol resistance gene was should be checked again.
They adjusted the concentration of chloramphenicol based on this analysis and finally succeeded in culturing the target strain.
At the beginning of April, BNUZH-China contacted us and stated that their project intends to utilize BC for burn treatment, which is similar to our last year's project. And this year we also continued the application of BC in our project. We both participated in the Southern China Regional Meeting, which deepened our understanding of the projects of both sides and decided to establish partnership.
Due to the academic and epidemic situation, we conducted our first online communication meeting on July 14th. In the meeting, all the team shared their projects, BNUZH-China used E. coli as a chassis to degrade bacterial cellulose film by anaerobically initiating cellulase. We proposed that oxygen does not gradually decrease during the generation of wound healing. Therefore, we suggested them to try to use the light control system and to have an in-depth discussion on the safety of the live bacterial products and the future direction of cooperation around the project content.
We shared that the bacterial cellulose film produced by the bacterial emulsion was too thin in the regular meeting on July 31st. BNUZH-China suggested that we adjust the emulsion composition and try to increase the film production time. Subsequently, we changed the observation time from half an hour to an hour. From the observation, we found that the production of film increased.
The DNA fragment (bphs-PcdrA-FleN-FleQ-pSEVA331) cloning situation of our experiment was not good on August 14th. So, we asked BNUZH-China for help, and they analyzed the advantages and disadvantages of different ligation methods and recommended us to use gibson Assembly. Finally, we took their suggestion and successfully got the needed DNA fragments.
During our regular meeting on August 28th, BNUZH-China shared their experience about the building of wiki to us. They suggested that it is important for us to make the wiki as easy as possible for people to read it and we should adapt the wiki to tablet, etc. Their suggestions inspired us to build our wiki.
During regular meeting on August 28th, BNUZH-China members with modeling experience shared his modeling experience with us, such as modeling does not need to use all of our own experimental data, but requires the model to fit the experiment. This solved our doubts in modeling.
In the regular meeting on September 11th, we discussed with BNUZH-China some issues in the safety form that we have confusion, such as the organisms used in the project in Question 14, chemicals, the hazards to people in the parts, the hazardous chemicals used may be ignored.
In addition to the above, BNUZH-China was rigorous and extremely serious in preparing for the weekly meetings, which made us realize that our communication was not well prepared and that we needed to learn from the BNUZH-China and that we should plan well in advance. This also allowed us to improve the quality of communication with all teams.
We invited BNUZH-China to participate in the online science lecture "Enter the World of Synthetic Biology" on August 6th. They shared with the audience the application of synthetic biology in the conservation of cultural relics and the treatment of burns, which broadened the audience's impression of the application of synthetic biology.
After the science lecture, we also invited them to participate in five-team Meetup to share the project with our other collaborating teams. Their project required sterilization before using BC, and we discussed the method of sterilization, whether it could be sterilized by strong oxide hydrogen peroxide? Will long time high temperature cooking bacterial cellulose sterilization make cellulose denatured? Give them new ideas.
During the regular meeting on August 14th, we invited BNUZH-China to write Microbial Hymns in synthetic biology poetry to help even liberal arts students understand synthetic biology easily, and BNUZH-China wrote a total of three pieces, which received more positive comments.
In the regular meeting on August 7th, BNUZH-China shared with us the method to increase cellulose yield. We had also read a paper on enhancing cellulose yield by adding growth factors in our preliminary research, so we shared this paper with them.
At the beginning of the project, BNUZH-China learned that we had experiments on bacterial cellulose (BC) and used a different strain than they did. They wanted to get BC produced from the our chassis for their experiments. Due to the epidemic and the project schedule, we sent our bacterial cellulose to them on September 12th.
BNUZH-China needed cellulase for their experiments on September 10th, but they were short of funds for their late experiments and had limited purchasing capacity. We had cellulase in stock, so we sent it to them on September 12th to help them with their experiments.
The BNUZH-China failed repeatedly in the process of PCR amplification of CenA and Cex fragments. We proposed them to try using gradient PCR. They later used gradient PCR to find out the most suitable annealing temperature and tried landing PCR to improve the specificity of the products, and finally successfully amplified and sequenced them correctly.
BNUZH-China frequently experienced poor cell growth status and poor wall attachment at the beginning of cell experiments. They initially thought it was contamination, but after changing to a new medium and performing mycoplasma testing, they found that no contamination occurred. We suggested that it could also be the poor quality of fetal bovine serum. After replaced with a different brand of fetal bovine serum, they found the cell growth status was significantly better.
We met at the iGEM Online Meeting, hold by HBUT-China, on July 8th. During the meeting, we learned about ergothioneine, the main product of HBUT-China project, which is a new strong antioxidant applied in cosmetics and food, and has similarity with our project's GSH. Meanwhile, HBUT-China also expressed the idea of cooperation with us, so we decided to have further in-depth communication.
We had another online communication with HBUT-China on July 14th, and we discussed common allergy problems in cosmetics in the project design, which inspired us to conduct in-depth research on allergy problems in GSH.
In addition, since HBUT-China had a two-year break in participation, they were confused about the rules of the competition, so our team captain Zhibing Mai, who has a lot of experience in participation, shared her experience about the competition, team management, and answered the details of participation. Based on the willingness to improve together, we established a partnership with HBUT-China and set up a WeChat group to plan weekly meetings for project progress sharing and cooperation discussions.
We invited HBUT-China to participate in the online science lecture "Enter the World of Synthetic Biology" on August 6th. During the meeting, they shared the inspiration of their project and the application of synthetic biology in synthetic raw materials, which broadened the audience's horizon.
After the lecture, They were invited to participate in five-team Meetup to help them get more feedback on their project.
We invited them on July 14th to co-produce a Chinese-English translation of synthetic biology-related vocabulary word book to reduce the difficulties in vocabulary for future Chinese iGEMers and thus better focus on synthetic biology, and we finally finalized the book on October 1st, collecting near 500 words together. In order to facilitate being used, we have also created both mobile app and web versions.
We expressed difficulties in modeling in the regular meeting on August 11th. HBUT-China shared with us what they learned from other teams in modeling, such as related growth prediction models for projects, gene pathway expression models. HBUT-China provided us with two ideas in the regular meeting on August 25th: one is the model of impact factor and protein binding, and the other is the model of cell and drug binding. They also suggested us to start by finding relevant literature and shared with us the modeling software "design expert". We referred to these excellent suggestions for preliminary modeling planning.
On August 6th, HBUT also helped us create a poster for the "Enter the World of Synthetic Biology" lecture.
HBUT-China invited us to participate in a rural painting education activity on August 11th. They went to Guangxi Dahua Yao Autonomous Region to educate them about synthetic biology painting, and we provided them with the materials needed for painting.
HBUT-China helped us design the web and APP interface of the word book.
During the 2022 iGEM Online Meetup on July 16th, we learned that NEFU_China's project is an efficient DNA assembly strategy that can control the product to reach the target yield. Our GSH production module also needs to control the yield of GSH to reach the desired value. This became an important reason for our partnership.
We had our first online communication on the evening of July 17th. During the meeting, we had an in-depth discussion on the design of our project, such as is G. hansenii a safe chassis? NEFU_China also shared with us the use of hydrogen peroxide stress to induce the cultivation of high antioxidant yielding strains. It was very rewarding for us. After discussing the collaboration plan, we decided to form a partnership relationship and set up a working group.
NEFU_China posted a three-line poem on synthetic biology on WeChat on August 13th. Inspired by this, we wanted to pay tribute to the microorganisms that have made great contributions to synthetic biology through poetry, and to express our knowledge of science in a literary way and to make synthetic biology more accessible to liberal arts students. After discussing with NEFU_China, we started to write and solicit three issues of microbial hymns together.
Due to the low GSH production of our engineering G. hansenii, NEFU_China suggested us to find the main 3 factors affecting GSH production of G. hansenii by response surface approach and find its optimization conditions to construct a yield prediction model.
We invited NEFU_China to participate in the online science "Enter the World of Synthetic Biology" on August 6th. In the meeting, they shared the connection between synthetic biology and our daily life, and it was a wonderful lecture that brought the audience closer to synthetic biology.
After the lecture, We also invited them to the five-team Meetup, In the meeting, they shared the phenomenon of strain degradation after hydrogen peroxide stress-induced screening of high antioxidant-producing strains, which caused us to think about it.
On August 9th, NEFU_China encountered difficulties in their experiment and failed to find the cause for a long time, so they asked us for help. After we learned about it, we found two experts (Wenwen Xiao and Shengwei Fu from the Quantitative Center of the Institute of Synthesis, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences) to solve the problem for them. They finally solved the problem successfully by making improvements based on the analysis results of the experts.Please see below for details.
Our target product is red, after we introduced the exogenous synthesis pathway into the brewer's yeast genome, the bacteria that produce the target product will turn red, we coated the plate medium and found that at first there were red single colonies, slowly white strains grew on the red single colonies, what is the reason? Is it degraded or is the yeast single colony impure (not really a single colony)?
Naked eye observation sometimes has bias and you can try to use microscope for further observation.
It is also recommended to delineate the red colonies several times and sequence the promoter and RBS of the white colonies to see if they are mutated.
