Team:QHFZ-China/Human Practices
Integrated Human Practices
Content
Overview
Part 1: Problem Investigation
Part 2: Project Optimization
Part 3: Constant Feedback and Professional Guidance
Part 4: Public and Professional Engagement
Overview
While we designed our project, our advisor from QHFZ-China 2019 told us that the storage methods of engineered bacteria vastly limit their daily usages. By interviewing other previous iGEM teams and asking them to judge the feasibility of our storage methods, we realized that a numerous number of different iGEM teams and participants have experienced bacteria storage issues and believe our project may be useful to support theirs. Thus, we interviewed some professionals to certify the possibility of our design. During our experiments, we kept connection with several experts and visited two pharmaceutical companies to improve our experimental process and applications. Moreover, we shared our existing results with our instructor Mr. Zhang extremely constantly, which really allowed us to get a lot of inspiration on how we can better improve our project.
Part 1: Problem Investigation
1. Interview with QHFZ-China 2019
Form: Offline
Interviewees: Team members of QHFZ-China 2019
Key Points:
(1) The usage of uric acid detection bacteria is limited by the existing preservation method.
(2) Many engineered bacteria is invented by various iGEM teams, but most of them may can’t really let their bacteria play its function in daily life.
Our Response:
(1) We realized that the preservation method need to be improved. The daily usage of engineered bacteria is mainly limited by the preservation method.
(2) We connect with more iGEM teams to get more information about the usage of their bacteria.
(3) We tend to provide a new storage methods by using lyophilization.
(4) To further certify feasibility of project, we seminar with synthetic biology professionals.
Details:
We learned about the original issue of engineered bacteria storage through QHFZ-China 2019. While we were researching for our project, we looked at the project of QHFZ-China 2019. During this process, we realized that although their bacteria was effective, it can be hard to store it at room temperature for people to use. To further prove this issue, we interviewed their former team leader Li Tianhong, who told us that the issue of bacteria storage does exist and is actually quite harmful to the utilization of their bacteria in real life. Through researching on the products which were made of saccharomycetes, we found that almost all the products of saccharomycetes that we saw on the market were produced by lyophilization. During our research, we also got to understand that lyophilization could be separated into two parts, freezing and drying. With the drying process, the metabolism of bacteria is paused. Hence, we concluded that lyophilization may be a good solution for us to preserve the bacteria at room temperature through pausing metabolism.
2. Seminar with synthetic biology professionals to certify feasibility of project
Form: Online
Interviewees: Weizhao chen (Professor at Shenzhen University), Jiajia Liu (Professor at Chinese Academy of Sciences)
Key Points:
(1) Prof. Chen leaded us to focus on the differences and advantages between lyophilization method and the existing methods.
(2) Prof. Liu pointed out that compared with freezing environment, desiccation was more stressful to bacteria and while lyophilizing, we needed to think about a solution on how to better protect the bacteria in desiccation process.
Our Response:
(1) We searched more information about lyophilization, then gave feedback to Prof. Chen about why we choose lyophilization. Hence, Prof.Chen agreed with our method.
(2) We found that out of all the sea animals, the tardigrade is a micro-organism that has resistances to ultralow-temperature, vacuum, desiccation, radiation and so on. From a study that on 2017, we knew that a protein in the tardigrade called TDPs can be used to increase the survival rate of bacteria under desiccation environments.
(3) We tend to add TDPs during the freeze drying to protect the bacteria.
Details:
Therefore, we decided to create a new bacteria storage method which can store the bacteria under room temperature. Through researching on the products which were made of saccharomycetes, we found that almost all the products of saccharomycetes that we saw on the market were produced by lyophilization. During our research, we also got to understand that lyophilization could be separated into two parts——freezing and drying. With the drying process, the metabolism of bacteria is paused. Hence, we concluded that lyophilization may be a good solution for us to preserve the bacteria at room temperature through pausing metabolism. In order to certify the feasibility of this idea, we interviewed professor Weizhao Chen. He told us that the sand preservation method is widely used to store the bacteria at room temperature, and led us to focus on the differences and advantages of the lyophilization method. After that, we searched information about sand preservation, we realized that because of the usage of sand, the bacteria can be very easily contaminated. Thus, we gave feedback to Prof. Chen about the reason why we didn’t choose sand preservation and Prof. Chen agreed with our lyophilization method. We also interviewed Prof. Liu ( the professor at Chinese Academy of Sciences). She considered that the process of lyophilization may be too extreme for bacteria to survival under it. She also pointed out that compared with freezing environment, desiccation was more stressful to bacteria and while lyophilizing, we needed to think about a solution on how to better protect the bacteria in desiccation process. Afterwards, we found that out of all the sea animals, The tardigrade is a micro-organism that has resistances to Ultralow-temperature, Vacuum, Desiccation, Radiation and so on. Also from a study that on 2017, we knew that a protein in the tardigrade called TDPs can be used to increase the survival rate of bacteria under desiccation environments.
3. Interview with iGEM teams and a Sierra Leonean entrepreneur to analyze whether they have experienced issues in bacteria storage or not.
Form: Online and offline
Interviewees: BHSF (2020),BJ101HS (2020), KEYSTONE (2020), QHFZ-China (2019), ECUST_China (2018), ASTWS-China (2020), BNU-China (2019), BEAS_China (2019) and NEU_CHINA (2020), Shan Jiang, Yikai Bao, Guangyuan Song, Tianqi Zhang, Miatta Momoh (a non-profit organization that advocates for African and Chinese businesses/social cooperation)
Key Points:
(1) Miatta Momoh stated out that the electricity to support the storage of these bacteria under normal circumstances is definitely an issue in Africa.
(2) Miatta Momoh agreed that our method will be extremely beneficial for the usage of engineered bacteria.
(3) We explained our project to 10 iGEMteams and provided them with an idea of how our project could benefit theirs.
(4) We asked them to rate the level of benefit our project may provide for them and created a graph to show the results.
(5) We realized that the needed of professional equipment limited the usage of engineering bacteria and have hidden danger.
(6) From the communication with Shan Jiang, we gained some knowledge about the tardigrade and we had a better understanding about their project in 2017.
Our Response:
(1) By analyzing the data, we understood that many teams have experienced similar issues with QHFZ-2019.
(2) We’ll keep contacting with Miatta Momoh for the future application in Africa of our project.
(3) We reassure that our project is doable. Afterwards, we began our experiments and continued our human practices work at the same time.
Details:
We expected other teams to experience similar issues, we hoped to focus on this problem and try to solve it for them as well as thousands of other iGEM teams with our project. Therefore, we interviewed several teams who engineered bacteria to make sure the issue is common. These teams include: BHSF (2020),BJ101HS (2020), KEYSTONE (2020), QHFZ-China (2019), ECUST_China (2018), ASTWS-China (2020), BNU-China (2019), BEAS_China (2019) and NEU_CHINA (2020). During our interviews, we explained our project to them and provided them with an idea of how our project could benefit theirs. Then, we asked them to rate the level of benefit our project may provide for them and created a graph to show the results. Also, Through these interviews, we understood that many teams have experienced similar issues with QHFZ-China 2019. For instance, KEYSTONE (2020) wanted to create an engineered-bacteria that could degrade plastic waste. However, because their bacteria need to be put into the waste in order for it to function, it needs to be able to be stored in room temperature for it to be transported.
Therefore, we offered them the idea of storing their bacteria in powder form through lyophilization with the help of our project. They really liked the idea and gave it a 5 for being the most beneficial. Similarly, many other teams who experienced related issues also believed that our project will be extremely helpful for the public usages of their projects, which led us to believe that it could be a project that have a decent mass appeal.
Additionally, a current alternative for our lyophilization storage is the ultra-low temperature freezer. However, during our interview with JNU-2019, the team members claimed that while they were working on their project, the freezer in their lab malfunctioned and caused all their results to be unusable. This led us to believe that our project could solve this issue as well, since powdered bacteria cannot experience inactivation. JNU also agreed that our method may be able to effectively solve this issue and remained in contact with us throughout our project.
Also, after we decided on our goal, we interviewed Miatta Momoh, a Sierra Leonean entrepreneur who is the Co-founder and business/development manager of Kente and Silk, a non-profit organization that advocates for African and Chinese businesses/social cooperation. In our interview, we discussed the necessity of cold-chain transport of engineered bacteria in medical businesses as well as in people's everyday lives. She stated that there are not a lot of current biomedical utilizations of engineered bacteria that she knows of, but the electricity to support the storage of these bacteria under normal circumstances is definitely an issue in Sierra Leone. As we purposed our plan to eliminate the use of freezing bacteria storage, she agreed that it will be extremely beneficial for the usage of engineered bacteria and introduced us to some of her contacts at Tsinghua University for further information.
As the issue gradually becomes real and obvious, we decided to use lyophilization as the optimal method for room temperature storage of engineered bacteria. We plan to transfer TDPs into the bacteria to preserve it during lyophilization process so that we could provide a new storage method for engineering bacteria under room temperature.
Part 2: Project Optimization
4. Interview with expert on micro-organisms to solve the issue on why our result was unstable.
Form: Phone call
Interviewees: Dr. Shaojie Li (Professor at Chinese Academy of Sciences)
Key Points:
(1) The existing survival rate of lyophilization is less than 10%, so that if we are able to increase the survival rate of the engineered bacteria to 10% or higher, we will already be making a huge innovation.
(2) Almost all the bacteria we used in laboratory are capable for living under ultralow- temperature.
Our Response:
(1) We changed our view about the survival rate in our experiments.
(2) We re-analyzed our data and we found that the trend of survival rate in all experiments were unanimous, which means the survival rate of experimental group was higher than the no-treatment control group.
Details:
During our experiments, we found that the results between the lyophilization we did each time were very unstable and we were all concerned about it. We wondered about what may have caused this situation and how we can solve this problem so that our method can be used in real life.
To find the answer, we interviewed Dr. Li (Professor at Chinese Academy of Sciences) who is an expert on micro-organisms. We asked him about whether the bacteria species we choose is capable for lyophilization. He told us that almost all the bacteria we used in laboratory are capable for living under ultralow- temperature, which means they have some resistance of freezing. He pointed out that the existing survival rate of lyophilization is less than 10%. This means if we are able to increase the survival rate of the engineered bacteria to 10% or higher, we will already be making a huge innovation.
Hence, we changed our view about the survival rate in our experiments the we found that the compare the result in the same experiments, the trend of survival rate was stable.
However, as we re-analyzed our data, we discovered a new issue: the survival between each individual experiment had a distinct difference. This indicates that the results of our experiments remained unstable nonetheless.
5. Visit Youcare Pharmaceutical Group Co. to gain deeper understanding and find the answer for the question above.
Form: Offline
Interviewees: Mr. Chu and Mr. Zhao
Key Points:
(1) Mr. chu pointed out that the differences in operation methods, lyophilization machines, desiccation speed and many other external conditions are all potential causes of this issue.
(2) Mr.Chu told us that we should focus on the comparison in the same experiment.
(3) Visited the lyophilization production workshop.
Our Response:
(1) We tried our best to standardize the operation method by following the protocol rigorously when doing lyophilize each time.
(2) We focused on the comparison in same experiment. By taking this action, the data of our results has been more stable, so that we got ideal results in August and September.
Details:
To gain a deeper understanding of lyophilization and figured out the reason for the unstable results, we visited Youcare Pharmaceutical Group Co. to find a way to adjust our lyophilization conditions for more stable results. Mr. Chu told us that the differences in operation methods, lyophilization machines, desiccation speed and many other external conditions are all potential causes of this issue. Thus, we realized that we should focus on the comparison in the same experiment. After that, we tried our best to standardize the operation method by following the protocol rigorously when doing lyophilize each time. Additionally, we realized that we should focus on the comparison of variables in the same experiment. After taking this action, our results have been more stable, and we retrieved ideal conclusions in August and September.
6. Interview with a biological resistance expert to explore the protection abilities of our proteins to increase the survival rate based on the existing situation.
Form: Phone call
Interviewee: Dr. Zhizhong Gong (Professor at Chinese Agricultural University)
Key Points:
(1) Dr. Gong pointed out that the combination of proteins may have resulted in resistance under extreme dry or cold conditions.
(2) Dr. Gong agreed with our bacteria preservation method. He also explained that the reason why some life have resistance is that many genes play similar roles to provide resistance simultaneously.
Our Response:
(1) We decided to combine different TDPs and transfer them into the bacteria to test whether combinations could improve the survival rate of bacteria.
(2) We tried to connect someone who is specialized in proteins to helpe us find the capable TDPs.
Details:
During our experiments we found that, after lyophilization, the survival rate of bacteria increased if we added single protein. But the effect was still limited. We wanted to improve its effect based on the protein we already had. In order to learn more about resistant, we contacted Dr. Gong (Professor at Chinese Agricultural University) who is an expert in biological resistance. We made a phone call with him, he agreed with our new bacteria preservation method. Most importantly, he told us that the reason why organisms have stress resistance is that there could be more than one gene playing its role in it, so that a combination of proteins may have resulted in resistance under extreme dry or cold conditions. Hence, we decided to combine different TDPs then transferred into the bacteria to tested whether it could improve the survival rate of bacteria.
7. Second interview with a previous iGEM leader who specializes in structural biology
Form: Online
Interviewee: Guangyuan Song
Key Points:
Guangyuan Song said that the proteins which have the most different structures may cover more functions of resistance during lyophilization.
Our Response:
(1) We used swiss model to predict the structure of all the TDPs we had. (2) We choose CAHS 106094 and SAHS 33020 for combination to further enhance the survival rate. More information is on Protein page.
Details:
To identified which two protein combinations are more likely to improve survival rate, we second interview with a previous iGEM leader who specializes in structural biology. Guangyuan Song helped us choose a pair of proteins that have the structure we need to achieve lyophilization. With his help, we choose CAHS 106094 and SAHS 33020, combined them on our plasmid. After lyophilized and stored under room temperature for 10 days, we analyzed the results and it shows that the combination of TDPs truly has the ability to give a better protectant to our bacteria. This result verified the suggestion that Dr.Gong gave to us and it provided a more realistic choice for our lyophilization experiment.
Part 3: Constant Feedback and Professional Guidance
We kept regularly contact with our instructor Mr. Zhang.
8. During the designing process
Key Points:
(1) Mr. Zhang helped us to evaluate the feasibility of our lyophilization method.
(2) He led us to think about how our project will benefit the society.
Our Response:
(1) We searched for various information about lyophilization in previous iGEM teams’ wiki and literature.
(2) We decided to use room temperature storage of engineered bacteria as our main target which we thought would widely benefit the society, as shown from the information we got in human practices.
9. During the experiments, we found that if we just added the TDPs which we bought from the synthesis company, the effect wasn’t idealized.
Key Points:
(1) He pointed out that we should try to control the expression of TDPs.
(2) Our experiments should do the quantitative analysis.
Our Response:
(1) We found different promoters with different intensity on iGEM’s parts page. Therefore, we choose J23100, J23107 and J23109.
(2) After we changed the T7 promoter with J23100, J23107 and J23109, the survival rate of bacteria displayed a gradient changing trend.
Details:
We discussed with our instructor, he pointed out that we should try to control the expression of TDPs, if the protein at highly expression could play its role in lyophilize, then it proved that TDPs truly could be a great protectants. Taking this suggestion, we found different promoters with different intensity on iGEM’s parts page. We choose J23100, J23107 and J23109 because we noticed the gradient between them, it shown that J23100 is a strong promoter, and J23107 as a medium promoter, J23109 as inferior promoter. Hence, if we combined it with the TDPs that we used, it would attained the target of controlling different expression of TDPs. After we transfer the J23100/107/109 promoter and TDPs combination into BL21, the result showed that with highly or middle expression, the TDPs elaborated its function. So why the previous effects wasn’t idealized was that the protein wasn’t obtained a suitable expression. And also the survival rate of bacteria appeared a gradient changing trend.
10. As we got some positive results, we wanted to verified an assume of room temperature storage.
Key Points:
(1) He suggested that after lyophilzation we should try conduct a room temperature storage for over 5 days.
(2) He told us that if the result is positive, it will identify that our storage method is useful and that it will go into operation in the future.
Our Response:
(1) We stored our lyophilization products under room temperature for 10 days, then observe the clones on LB plates. The results showed that our assumption is right.
(2) We considered transferring our proteins into an engineering bacteria designed by iGEM teams from this year to know whether our project really gave out a new storage methods. We connected with NEFU-China.
11. More improvements we can do
Key Points:
Mr.Zhang told us to pay attention to the degradation of TDPs, so that it would avoid the surplus stress for bacteria.
Our Response:
(1) To reduce the possibility of this issue, we decided to add a protein degradation tag.
(2) We did some research then we found that mf-lon and AAV tag may be useful.
Details:
Mr.Zhang also told us to pay attention to the degradation of TDPs, because as for the bacteria which elaborated its function, the TDPs are surplus for the bacteria and if it still expressed in the bacteria, it will cause more burden, also the surplus expression of TDPs will affect the original function of bacteria. To reduce the possibility of this issue, we decided to add a protein degradation tag. During our research, we found that mf-lon and AAV tag may be useful.
But with the limitation of time, we could just predicted this result by using mathematic modeling, more information is shown on modeling page. We hope to construct the plasmid with degradation tag, by using the technology called western, we could confirm whether the tag is useful.
Part 4: Public And Professional Engagement
12. Visit Tide Pharmaceutical Co.
Form: Offline
Interviewees: The CEO of Tide, Mr. Xie and Executive, Mr. Lu
Key Points:
(1) Mr.Lu explained how lyophilization appear in different parts of their machines so that we could made an analogy on the mechanism between our instrument and theirs.
(2) He also gave us an important suggestion that we need to control the concentration of glucose between 3% to 15%.
(3) Mr. Xie showed us the development of the company and a variety of medicines they made using lyophilization
Our Response:
(1) We decided to use 3% glucose during our experiments.
(2) We went to the production workshop to see how different types of lyophilization machine worked.
Details:
We visited Visit Tide Pharmaceutical Co., the biggest pharmaceutical company in China to get advice for lyophilization. The CEO of Tide, Mr. Xie and Executive, Mr. Lu gave us some examples and introduction of lyophilization. We went to the production workshop to see how different types of lyophilization machines worked. Following the production line, we saw the final product of lyophilization and observed its shapes. While visiting the production line, Mr.Lu explained how the lyophilization happened in different parts of machine so that we could make an analogy on the mechanism between our instrument and theirs. Afterwards, as we discussed in the meeting room, Mr. Xie showed us the development of the company and a variety of medicines they made by using lyophilization. Mr.Lu introduced different forms of lyophilization protectants such as Glucose or non-fat milk powder. We told him that we wanted to use glucose as the protectant. To explain why we will need it, he told us that the reason why we need protectant(s) is to protect the bacteria in freezing process because ice crystals will penetrate the cell membrane, destroying the cells. He also gave us an important suggestion that we need to control the concentration of glucose between 3% and 15%
13. Follow-up interview with Professor Weizhao Chen
Form: Online
Interviewee: Weizhao Chen (Professor at Shenzhen University)
Key Points:
He guided us to think about what product we want to present and how our product really exercised its usage.
Our Response:
(1) We took his suggestion and thought about our proof of concept.
(2) We shared our experimental processes with him.
Details:
We made follow-up Interview with Dr. Chen (Professor at Shenzhen University). We shared our experimental process and our feeling with him. He guided us to think about what product we want to presented and how our product really exercised its usage. We took this suggestion and after discussion, we decided to build a platform for the user. By analysing the information that the user gives to us, our platform will select the best methods for making the product that the user wants. So this became our proof of concept and we also made detailed explanations in this page.
Last but not least, we held monthly national iGEMer Meetups from August to October and took part in Northeastern Coastal Meetup. We got to understand each other’s projects and provided each other with criticisms. We found out that many teams may need our storage (potential interviewees/collaborators). What's more, we advised each other about the projects and provided each other with useful feedback. We met with one of our biggest collaborators—NEFU through this meetup. To get more information, click here to get to the collaboration page. Moreover, during our second monthly meet up, we got to understand more issues each team was facing, provided each other with advice, and even collaborated with Keystone Academy to host an iGEM synthetic biology event at our own school (See education for details) to resolve our issues together.
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