HUMAN PRACTICES
Partnership
The collaboration with ShanghaiTech_China runs through our project, and the common goal that we both work towards is not only the vision of space but also the future expectations of food production.
At the initial stage of the project, the two teams hit it off and appreciated each other's projects.
When determining the project direction, ShanghaiTech_China learned that BUCT-China uses E. coli to produce edible products - cultured meat, so they changed the chassis bacteria from spirulina to E. coli which preserves the production of food and has greatly improved by synthetic biology. At the same time, BUCT-China's cultured meat food can be used as a downstream industry to land the project.
BUCT-China also knows that our two teams can utilize our chassis bacteria as platforms for each other, and ShanghaiTech_China is our vision. Keeping our eyes on the stars while with both feet on the ground. Since then, taking space exploration and interstellar migration as the background of our grand vision has been firmly established. We and ShanghaiTech_China contacted each other and conducted preliminary exchanges and discussions. After introducing our respective inspirations and preliminary plans, we put forward each team’s opinions on the overall idea together.
However, in the early and middle stages of the project, Shanghai suffered from COVID-19 and was therefore subject to lockdown control. ShanghaiTech_China’s projects had been affected a lot which is very difficult to develop the experimental part furtherly. BUCT-China was also in quarantine and lockdown for a long time in the middle and last stages of the project due to the impact of the Beijing epidemic, so it was unable to carry out experiments. However, COVID-19 has not knocked us down but allowed our relationships to get closer, and we can support each other through cooperation.
It is not only the support of concepts but also our mutual help in experiments. When the progress of the experiment was limited by the epidemic, we worked together to tap stakeholders and conduct research, and jointly publicized the knowledge of synthetic biology. (modeling)
the experiment design of BUCT-China offered inspiration to ShanghaiTech_China who adjusted their project plan and initially established the project track. Due to the impact of the epidemic, we held online meetings to better communicate. The two teams sincerely support each other in concept and discussion on experimental design. We arranged meetings together frequently in order to have a further understanding which allows us to provide some valuable advice for both teams.
ShanghaiTech_China asked us about the suggestion of using E. coli as chassis to produce food (cell-cultured meat), and we provided theoretical and experimental support to them.
In addition, we have maintained continuous contact with every team member.
Experiment
quorum sensing system with an alternate expression of the idea
ShanghaiTech_China suggested that we could design a system that can express alternately
In the communication with the students in the experimental design group, we proposed the degradation label to ShanghaiTech_China students gave inspiration.
Experimental design: insect catching and supplement, joint efforts to tap stakeholders (questionnaires), joint publicity
Industrialization:
mussel protein: this method can increase adhesion
BUCT-China proposed that it may be necessary to improve cell adhesion in current cell meat production. ShanghaiTech_China, based on the experience of past projects, told us that mussel mucins have strong adhesion which is completely harmless and biodegradable, so maybe mfp-5 can be used to promote the adhesion of cell meat. As a result, we decided to cooperate with Synmeta (Shanghai Yiru Technology Co., Ltd.), a synthetic biology company derived from the iGEM competition of ShanghaiTech_China, to develop cell meat and solve the adhesion problem in cell meat synthesis.
At the same time, we had an in-depth exchange of microsphere culture methods. ShanghaiTech_China uses microspheres to cultivate microorganisms to achieve the purpose of co-culture. We also use microspheres to conduct cell culture in order to form the final meat. In the process of communication, both of us have contributed a lot to each other's design.
Experiment
Shanghai_ Tech proposed that it is necessary to show that the three chassis bacteria have different fluorescence, and the fluorescence brightness is insufficient
We send the sequence of the fluorescent protein to ShanghaiTech_China. The fluorescent protein has high brightness and is red. It can be compared with E.coli (blue) and cyanobacteria (green) to identify after their practice.
HP
Meanwhile, the events we hold together benefit us, including joining scientific education and designing the questionnaire.
We attended the Science Lecture (metabolism) held by ShanghaiTech_China
Collaboration questionnaire with ShanghaiTech_China The questionnaire “Just the right question of the thing we care about” is mainly focused on the public awareness and recognition of cultured meat and the Mini Bioproduction Circle System on Mars base (MBCS). The content includes the food safety problem and Mars immigration plans which offers us the requirement and feedback for our project.
(experimental support, modeling, and joint efforts to explore stakeholders)
After a half year of mutual running in and help, we successfully solved many technical problems in the experimental practice. Through our joint efforts, both teams have made great progress in their projects. We held a further meeting for consultation and confirmed the cooperation content in the final stage.
The two teams conducted preliminary cooperation on some aspects of human practice at the previous meeting and jointly explored common stakeholders. Therefore, when designing social practice, we mainly communicate from laboratory to industrial production, food safety, industrialization, and other aspects, and contacted Mengniu Xidong Liu, to conduct interviews for exchanges. Our projects have also been fully recognized.
In the later stage, the experiments of both sides have basically reached the final stage, and some experimental results have been achieved. Through the private communication of the students during the experiment and the convening of the meeting, we put forward the difficulties encountered in the experiment one by one and exchanged views to help each other.
Experiment
Due to the lockdown control of COVID-19, our experimental progress has been greatly affected. Therefore, we sent the three synthetic plasmids to ShanghaiTech_China, and they helped us measure the fluorescent expression content, and alternate expression was exhibited using fluorescence intensity. In addition, students from ShanghaiTech_China found that the activity and multiple carbon source response promoter were not enough, and those commonly used in eukaryotes were not applicable to prokaryotes. After the communication and discussion with us, they found that the delta-activator can be used in their design, so they take the activator in our plasmid to practice, and analyzed the fluorescent expression through the design below.
ShanghaiTech_China: In our project, we choose starvation promoters for the nutrient response. However, after two cycles of optimizations, we only get a maximum fold-change of 8.2, which is still not enough for complex genetic circuit design. Some strategies like amplifier (Figure 1A) or positive feedback (Figure 1B) have been used in eukaryotic systems.[1] These strategies are not well developed in prokaryotic systems because of the lack of transcription activators. After communicating with BUCT-China, we learned that an activator delta from phi R73 phage can bind to a specific sequence on DNA and activate a RpoD-dependent transcription. Based on the two strategies in the figure below, we constructed seven plasmids with our starvation promoters, PcstA variants.
[1] Hicks, M., Bachmann, T.T., and Wang, B. (2020). Synthetic Biology Enables Programmable CellBased Biosensors. Chemphyschem 21, 131. 10.1002/cphc.201901191.
We attempted to use tetR and p(tetR) to participate in the negative feedback loop that constitutes the gene oscillator and construct the following plasmid.
The plasmid designed by us
ShanghaiTech_China has helped us to measure the expression of EGFP controlled by the p(tetR) promoter under gradient concentrations of tetracycline.
Expression of EGFP controlled by the p(tetR) promoter under gradient concentrations of tetracycline
Tetracycline turned on p (TetR), GFP was expressed, and the bacterial solution was green
ShanghaiTech_China has helped us prove that the TeTR-P (TetR) pair meets theoretical expectations and can repress and de-repress normally.
Meanwhile, ShanghaiTech_China hopes to degrade AHL in order to reduce the leakage expression in nitrogen source response. As a result, we discussed that we can add a degrading enzyme to AHL, and a repressor protein with a promoter can be tried. Therefore, a set of promoters P- λ And CI repressor from our experiment design were sent to ShanghaiTech_China to practice.
ShanghaiTech_China hopes to verify that E.coli of Sac+ secretes acetate when growing in a sucrose culture medium. We analyzed the supernatant for them and detected whether acetate is produced, with the help of chemical analysis.
In addition, we exchanged the chassis bacteria. For ShanghaiTech_China, our E.coli which can produce edible substances, is the downstream industry of the other project, helping the team integrate with the practice and production of the industrialized food industry. They also sent their blue-green algae to us, hoping to integrate photosynthesis into our project. From photosynthesis to PHFA, this is in line with the current environmental protection issues and reduction of carbon emissions. These are our first steps toward the future, showing bright prospects.
HP
ShanghaiTech_China lacks information from the industry, so they hoped that we can help find the opinions and information of stakeholders they have not interviewed. We provided help, found Liu Xidong, a senior engineer of Mengniu, a food enterprise, and conducted an interview with ShanghaiTech_China, gaining valuable opinions. (See the interview section)
The well-coordination has a great impact on both teams which improves our projects from many stages. Our aims offer a subtle blend of our projects which definitely establish the grand prospect from earth to space, and we all looking forward to having the sincere cooperation continuing in the future.
In last year's iGEM competition, BIT-China and BUCT-China conducted close cooperation as partners in the whole stage and various fields of the competition. Both teams learned a lot from the in-depth communications on various aspects. What’s more, both BIT and BUCT are located in Beijing, which makes it possible to cooperate with each other face to face under the strict local epidemic prevention restrictions. Therefore, after exchanging project ideas this year, the two teams decided to carry out long-term cooperation again.
During the communications, we acknowledged that in order to improve the efficiency of their bio-hydrogenizes system, BIT-China is desired to generate a series of oxygen tolerant enzymes by mutation breeding. Meanwhile, they also have great interest in algae, asking whether it is a good choice for conducting their hydrogenizes system. After in-depth communications, we point out that focusing on mutagenesis does not reflect the role of synthetic biology, therefore not a good solution. What’s more, algae are not commonly used in synthetic biology, the genetic operation is more complex and lacks certain experience, which may bring some difficulty to the experiment. Hence, we recommend they use some common model strains to conduct their future experiments.
BIT-China wanted to know whether biological hydrogen production and nitrogen fixation have advantages over traditional industrial operations. With our advisers’ suggestions, we propose that compared to chemical biological hydrogen production or nitrogen fixation technologies, biological productions are relatively immature, which will be more costly. However, it is highly environmentally friendly, which can reduce greenhouse gas emissions besides less energy consumption. Hence, it has a certain potential for future utilization.
BIT-China team wanted to construct a hypoxic environment, in which the concentration of oxygen inside the cell was tested. We discussed the methods for testing intracellular oxygen concentration, suggesting that a promoter with high activity under low oxygen can be selected to reflect the oxygen concentration, followed by reporter genes such as fluorescent proteins.
BIT-China has considered whether the low-oxygen environment benefits production or not. In this regard, we also put forward some ideas. For example, hydrogenase or nitrogenase can be further introduced into the recombinant bacteria, and the same enzyme can be introduced into the conventional E. coli in order to compare and test whether the construction of the hypoxic device really plays a role.
When we select the appropriate elements to construct the feedback loop of the gene oscillator, BIT-China suggests that lactose operon or tetracycline operon can be used as the inhibitory control. For promoting elements, AHL can promote the expression of some post-promoter genes. The lactose operon can be properly modified or combined with other elements to achieve well-gene oscillation. In addition, since gene oscillators widely exist in quorum sensing, adding this unique mechanism can be taken into consideration. Apart from that, the oscillation effect can be measured according to the experiment, such as whether the oscillation period is appropriate, whether it has good robustness, and so on.
Knowing that we need to observe the fluorescence intensity of engineering strains periodically, BIT-China suggests that the microplate reader can be used for detection. The first thing is to know what kind of fluorescence it is, and its wavelength. After that, a microplate reader can be utilized to measure the absorbance value, and a specific wavelength can be fixed to measure the intensity of the emitted light and the fluorescence intensity.
Both BIT and we found that it is difficult to connect our genes in our discussion. The soybean hemoglobin gene of BIT-China comes from the soybean genome, which is of eukaryotic origin. It is also challenging to optimize the intron optimization, codon optimization, and our team's other elements. And the phage sources are not stored in our laboratory. After the discussion, we believe that if the traditional splicing method is too heavy a workload, the gene synthesis method can be used to build more complex elements. After that, we all adopted the gene synthesis method, which significantly improved the experimental efficiency.
When selecting the inducible promoter, BIT-China adopts the lactose operon on the plasmid vector, which requires IPTG to induce expression. Because the regulation gene expression system, which has rich characterization data, is commonly used. According to the experimental scheme, we suggest using TetR and corresponding p(TetR), which called to be tetracycline operon control. And LacI and Lac inhibition promoter has a similar effect. The Tet system’s corresponding inducer is tetracycline, and it has the characteristics of low background and high induction multiple.
In the absence of induction, the target gene has a low expression level, and its expression level will be increased after the induction. The activity after the induction can reach 10,000 times more than before. But a small amount is enough because tetracycline is an antibiotic and has an inhibitory effect on bacteria. The BUCT-China will send to the plasmid corresponding to the BIT-China, containing the TetR gene and the p(TetR) promoter. Both parties tried accordingly.
The application of bean hemoglobin: We need to consider how to ensure complete contact between cells and oxygen and nutrients. BIT-China has noticed this and recommended the bean hemoglobin that they are preparing to express. In the production of cultured meat, product whitening is a common problem. In addition, the chicken cells we used are relatively pale compared with the muscle cells of mammals such as pigs, cattle, and sheep. In the initial production process, our product color is not ideal, and we have been looking for feasible ways to improve them. After a thorough discussion with BIT-China, we decided to add bean hemoglobin to the product to improve the color of the product and to give consumers a better impression. Soybean hemoglobin is a commonly used food additive in cultured meat products, which can significantly improve the taste, flavor, and color authenticity of cultured meat products. After a discussion with the BIT-China, we envisioned the addition of hemoglobin to the microsphere material. The scheme is easy to operate and control and gives the product an excellent natural color after forming. Adding bean hemoglobin to the microsphere can effectively improve the oxygen concentration in the surrounding environment of the cell culture stage, promoting cell growth and its metabolism, and also has a specific buffer effect to prevent the excessive fluctuation of oxygen concentration from causing adverse effects on cell growth. We also plan to reach a stable supply relationship on soybean hemoglobin in the future process of entrepreneurship.
We also provide BIT-China with a reporter gene (mRFP1) that has excellent performance. Its advantage is that it does not need to be excited by ultraviolet light and only needs normal light to be excited to produce red fluorescence, which is easy to operate. The plasmid we sent to BIT China also provided the document of gene sequence for BIT China to try.
BIT-China: In order to provide sufficient data support for modeling, we plan to cultivate the reconstituted strains in groups. Each group will use IPTG inducer of gradient concentration to induce expression and observe the expression at regular intervals. Do you have relevant experience and suggestions?
BUCT-China: IPTG has high induction efficiency, and it usually requires only a small amount (1mmol/L) to achieve the ideal induction effect. We propose to design a series of concentration gradients around 1mmol/L. In addition, the induction time of IPTG also has an impact on the experimental results. Induction in the early stage of E. coli growth may increase the metabolic burden, leading to the final growth of bacteria is not very vigorous. The logarithmic late induction may avoid the metabolic burden of foreign proteins on bacteria, and improper induction time may also lead to the formation of inclusion bodies. Therefore, we suggest that you also explore an appropriate induction time. At the same time, in order to facilitate the operation of the experiment, we suggest using the porous plate matched with the microplate reader as shown in the figure to cultivate engineering bacteria, so that only the whole plate is put into the microplate reader for measurement at regular intervals.
BIT-China participated in the online multi-university joint science popularization held by BUCT-China
In the middle of August, BUCT China held a two-day online multi-university joint science popularization lecture. Before the science popularization speech, the two sides exchanged views and supported each other in the form of activities and other details. BIT-China and BUCT-China belong to the group of energy utilization and environmental protection. On this platform where they can communicate with each other and seek cooperation opportunities, the two teams take this opportunity to exchange the existing results of various parts and show the cooperation results to other participants.
BIT students come to visit BUCT
Design of the United Badge
In order to better publicize the project of the two teams, the students from the art teams of both sides designed the united team emblem together. The main body of the team logo is the mascots of the project of the two teams: E.coli of BIT-China and cultured meat of BUCT-China. Both sides used the team logo in the later joint activities or discussions to promote cooperation between the two teams.
Peripheral design and distribution
In order to better publicize the cooperation between the two teams and the iGEM competition to the public, the art teams of both sides choose masks as the joint periphery in combination with the epidemic background.
In the modeling part, the two teams conducted in-depth communication and discussion on modeling, simulation, calculation, and other methods, and jointly learned and improved.
Peripheral design and distribution
The two teams discussed how to determine and predict the parameters of the model and jointly decided to use Monte Carlo, literature survey, and other methods in the subsequent modeling process.
The two teams discussed the construction of ode ordinary differential equations and analyzed the assumptions of model equations, further practical methods, and improvements.
Peripheral design and distribution
BUCT-China→BIT-China：
In the process of dynamic simulation involving the construction of gene circuits, BIT-China took our suggestions and learned the simbiology toolbox. They found the gene circuits and gene elements in the project, in order to establish and run a dynamic model for the project, so as to achieve the purpose of simulating the dynamic process of the project.
BIT-China→BUCT-China：
When it comes to the evaluation of promoter strength and prediction of the certain performance of PHFA, BIT-China suggests that we consider using the neural network prediction method to predict some values that are difficult to measure directly when the data volume is sufficient. In addition, BIT China provides us with relevant BP neural network prediction codes and toolkits.
Since BIT-China's project is based on oxygen diffusion and consumption and differential equations with physical deduction, our project is to model the growth of cells in the microsphere system. Therefore, we learned the idea that BIT China can learn from physics, so as to build more sophisticated cell culture and consumption models (such as mechanical analysis of cell adhesion, nutrient diffusion, and consumption).
Later stage
After the modeling is basically completed, the two teams communicated with each other about the model defects and deduced the equation. We checked and discussed the existing contents to make the model established more rigorous and realistic.
We have also come to a consensus on the likelihood of further progress. BIT-China produces hydrogen and fixes nitrogen to support renewable energy or environmental protection. Its objective is to efficiently use biological processes to produce goods for people while protecting the environment, maintaining water supplies, and reducing pollution from industrial manufacturing. By substituting our cultured meat for meat from animals, we can lessen the demand for additional land and the harm that animal husbandry causes to the environment. Additionally, it can effectively preserve water resources, which are crucial for protecting the environment. The two parties will look into novel ways to use biology to protect the environment, adopt environmental preservation as a direction for collaboration, and save resources.
A hypoxia system was created by BIT-China. By utilizing their hypoxic system, it is anticipated that the produced compounds will have more stability and that some metabolic pathways will be more active and selective, increasing production efficiency, which is essential for future research and development. By using the promotion and inhibition relationship between various biological elements, BIT-China plans to develop a system in the future to realize the controllable change of intracellular oxygen concentration with time and the external environment, i.e., to make full use of oxygen. This system design is inspired by the gene oscillator we developed.