Team:iBowu-China/Human Practices
Human Practices
Overview of Human Practices Activities
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Stakeholder
7 Visits -
Experts
5 Interviews -
IGEM Teams
5 Events
Overview: Perspective on Human Practices from iBowu-China 2021
Human practice especially integrated human practice is intended to parallelly support the development of our IGEM project either in lab or out, possessing its significance in project design and lab operations. Thus, including two main elements – pitching of our project and receiving expertized feedback. In order to make the best out of integrated human practice this year, we experienced scrupulous planning and sufficient interviews with herb doctors, stakeholders/potential users, and professional TCM practitioners/experts. To further improve and modify our project, we invested in a profusion of time, effort, and resources, paying visits to listed TCM factories and centennial TCM stores/hospitals; obtaining opportunities to deepen our knowledge on TCM and receiving structural feedback from specialists. Overall, providing key information to better shape our project, preventing us from taking detours on the way.
Stage 1: Project Ideation
Interview with Greatbay_SCIE 2019
Form: Online, phone chat.
Keypoints: Integrated Human Practices could start in the ideation stage and cover the entire project.
Our responses:
We started human practices while we brainstorm for project by researching literatures, talking to iGEMers and interviewing specialists.
Details:
Suggestions on experiments: 1) Plan your time and start early. 2) When experiments fail mostly due to skill problems - repeat experiments 3）If result and hypothesis don't match - some variables may bring other new ideas.
Suggestions on HP: 1) Integrated HP runs through the entire project. Example: they visited ancient method of refining indigo blue - providing some of the hidden scientific principles, such as what chemicals are needed to change color. 2) Encourage the members to do what they are interested in.
Suggestions on Public engagement: 1) Create original materials - such as documentary they created introduced to traditional craftsmen who don't accept new technologies (because of their work) 2) Advice for video production: Consider your audience - host some offline experiences
Feedback from 4/18 Meetup helped with ideation
For the hosting of the local meetup in the Beijing area, one can see our collaboration page.
On the meetup, we introduced a few of our project proposals, including manufacturing an anti-depression substance called psilocybin, building a heavy-metal detection toolbox, and synthesizing an herbal extract commonly found in traditional Chinese medicine. Dr. Li Cheng from CCiC commented on the safety aspect and ruled out the first idea since psilocybin is a strictly controlled substance that had severe safety hazards. The detection toolbox was of great value, but his experience indicated the project would be really hard to finish within the time limit of the iGEM season. Therefore we started to look further into the herbal extract synthesizing project idea.
Interview with Prof. Chunnian He at The Institute of Medicinal Plant Development (IMPLAD)
Form: In person visits.
Keypoints:
1. Only the herbal medicine grown in certain areas can reach a sufficient effectiveness.
2. Licorice is of great pharmaceutical value and great environmental value as well.
3. A synbio-pathway using herbal extracts increases drug safety.
Our responses:
We picked licorice as our target herbal medicine, and later in the literature, we found its effective ingredient is glycyrrhetinic acid (GA). [Ref 3, 4] Because a biological pathway for its synthesis will help the patients to acquire cheaper medications, and also help protect the environment.
The Institute of Medicinal Plant Development (IMPLAD)
Prof. He answered our questions.
Prof. He introduced the institute to us.
Details
Prof. Chunnian He works at The Institute of Medicinal Plant Development (IMPLAD) which is an affiliated national research institute with the Chinese Academy of Medical Sciences. Prof. He specializes in analyzing compounds found in herbal extracts whose chemical fingerprints have been identified before. He introduced the current status of herbal medicine resources, supply and demand and also the chemical methods used for analyzing and treating herbal medicine extracts.
Prof. He introduced the value of Licorice from his work experience. Licorice is widely used for pharmaceutical and food additives. Harvesting the licorice plant destroys the soil environment because the effective ingredients exist in licorice roots, which extend far and deep into the ground.
Prof. He introduced a new technology already developed by their institue: a software that simulates plant growth environment (adaptive analysis system of Chinese herbal medicine). He encouraged us to learn more about the growth environment of the herb we wanted to study.
Prof. He talked about a few herbs who could be valuable research objects, while licorice was one of them. Another interesting herb was saffron glycoside that is proven to be effective against liver disease/tumor. The substance's host herb is very rare to find.
Prof. He talked about the safety aspect of Chinese medicine extract. For toxicology studies, there are different types: acute toxicity/chronic toxicity, genotoxicity (for reproduction), trigenesity (carcinogenic/teratogenic/mutagenic). It is important to learn the toxicity of a herb but due to complexity in its ingredients, the study is usually difficult. What are the advantages of synthetic herbal extract: their toxicity is easy to study due to its simplicity.
Stage 2: Project Design
Interview with Stakeholder Hosiptal
Hospitals are potential stakeholders who can make use of the herbal extract, and in our case glycyrrhetinic acid. We visited Kuanjie Chinese Medicine Hospital, a state-operated hospital that treats a large population every day in Beijng, to learn from the Chief of Nurse about the needs of practitioners and patients.
We learned a big group in the population do not observe the correct usage for herbal medicine, which can cause harmful consequences. This inspired us to engage the public for better understanding of drug safety for herbal medicine, and later the idea turned into our documentary project for science communications
We also learned the current medical cost of using glycyrrhetinic acid is pretty high. Later after the visit, we found in the literature [6] and realized the cost was high because the conventional extraction methods had many disadvantages such as low yield, low purity and high environmental pressure.
Interview with Kangerfu Pharmaceutical Beijing
Form: In person visits.
What we learned:
1. The traditional processing workflow for extracting ingredients of traditional Chinese medicine.
2. The pharmaceutical companies' need for quality control in the produced glycyrrhetinic acid and other herbal extract.
Beijing Kangerfu Pharmaceutical Ltd.
Worker prepared herbal medicine for grounding.
Purification under high temperature and pressure.
Interview with Beijing IncreasePharma
Beijing IncreasePharma is a pharmaceutical company in developing and delivering both chemical drugs and chinese medicine drugs. They also host a drug safety research and test center.
We learned about the safety measures taken in chemical drug production and herbal drug product. This increased our confidence that if we succeeded with the product, GA can be safely manufactured and delivered to the end users.
6. Interview with TCM expert
Dr. Gao Feng from Beijing Century Maijin is a doctor specialized in treating chronic diseases with traditional chinese medicine and herbal extract. From the interview with him, we learned that using herbal extracts is welcomed by the doctors with caveats. GA as the herbal extract from licorice has been widely used as an auxillary medicine (which has been reported by a lot of research), and most of the GA has to be obtained through the traditional extract methods with their limitations and disadvantages. Another big problem is many herbal extracts or effective ingredients, even though can be extracted and separated from raw materials, can not be directly synthesized even in the traditional methods. They are sensitive to harsh synthesis conditions. Dr. Gao encouraged us to explore using synthetic biological methods to achieve the synthesis of the herbal extract that can not be produced in conventional methods yet.
Stage 3: Project Modifications
7. Interview with Prof. Lv
What we learned: the enzyme we wanted to use β-glucuronidase exists in many species, and the activity of the enzymes from different species could be quite different. Dr. Lv Bo suggested we also try other species, such as Aspergillus oryzae.
Our response: after literature search [Ref 4, 5], we selected β-glucuronidase from Aspergillus oryzae [4] and Thermotoga maritima [5], in addition to E. coli. Experiment results are presented on our wiki.
During our search for specialists to interview, we noticed an expert in the field of using enzymes for synthesizing chemical compounds including herbal extract, Dr. Lv Bo. He is located in Beijing and works as a professor at BIT, i.e, Beijing Institute of Technology. We first established a contact with the team leader of BIT-China in the QQ chatting group, and he kindly put us in contact with Prof. Lv.
Dr. Lv Bo was an iGEMer too! He kindly offered to hold an in-person meeting with our team in his office. We presented the project design of using β-glucuronidase to convert for glycyrrhetinic acid, which is the effective ingredient in licorice. Dr. Lv Bo had strong previous experience in this specific enzyme and suggested we look into the protein structure and try the enzyme from different species. The reason is that even though the basic functions are alike, the exact amino acid sequence, the folding, the spatial structure and potential interaction site would be different for enzymes from different species. From his own experience, he suggested Aspergillus oryzae beside E. coli.
8. Interview with Dr. Wang from a protein production pharmaceutical company
What we learned: Try different induction temperatures and try adding carbon sources and nitrogen sources into bacteria culture.
Our response: We made the plan according to the suggestions but did not have enough time for the experiments
What we learned: IPTG induction might raise the production cost due to the cost of IPTG. We should also consider constitutive promoters and test both cases.
Our response: We selected to test with a constitutive promoter J23101 (which we conincidentally acquired on the sfGFP plasmid sent by BNDS-China.)
We visited AIDE, a subsidiary of the Yibai Pharmaceutical Group. Dr. Jun Wang, the general manager of AIDE Pharmaceuticals, and a drug development engineer accepted our interview. Our project focused on protein production, and our partner, CKWA-China team's project focused on producing a protease for cancer treatment. Both are closely related to Dr. Wang's specialty. He would be both an expert in project improvement for us, and a potential stakeholder for us. We and CKWA-China shared this Human Practices interview and both learned a great deal from it. AIDE Pharmaceuticals's main product is rteplase for injection: recombinant human tissue-type fibrinogen kinase (rt-PA), a protein that is not easy to produce. Dr. Wang introduced to us that there is a very large gap between success in the lab and industrial mass production. A rare amount of inpurity may not affect lab production, but when accumulated in the industrial mass production, it may lead to mass production failure. We introduced our project design and the difficulties we had in our experiments and asked about how to increase the production yield. We presented our proposal to try different E. coli strains for induction and Dr. Wang agreed to our plan. He also suggested we consider different induction temperatures and try adding carbon sources and nitrogen sources into our bacteria culture. Dr. Wang educated us on the process after a lab production success. If we could complete a small-scale production and purification in the laboratory, our next step would be to have completed a mid-size production test on the scale of liter volume of bacteria culture. Then we needed to consider how to proceed to production on an industrial scale on the level of tons. Along with the topic of industrial production, he mentioned the considerations for cost control. He suggested that IPTG induction in industrial production could lift the production cost in some cases where the induced expression is not strong enough to produce large quantities of protein. He suggested we should also consider testing a constitutive promoter.
9. Interview with Prof. Li
What we learned:
Glycyrrhetinic acid can be detected by HPLC, but the method is not suitable for students due to high cost and long experiment time.
Enzyme activity can be detected with simpler method using a color-changing substance.
Our response: We changed our experiment plan and integrated Phenolphthalein β-D-glucuronide into the plan for detection.
Prof. Li Lina works at Beijing University of Chinese Medicine, and she specializes in the area of Chinese herbal medicine effect of pneumonia. She had research experience with licorice and glycyrrhetinic acid, and she grew very interested in our project in that glycyrrhetinic acid could be useful in treating cancer and inflamation and thus directly related to her work.
Prof. Li introduced to us that usually a compound like glycyrrhetinic acid can not be directly observed or detected using easy and quick experimental methods. HPLC is the method that biotech companies and university reseachers use for detection. However the usage of HPLC requires special training, the experiment would take a long period to finish with a highly costly consumption of special containers. This would not be a proper method for us students.
She suggested that to detect if the enzyme has activity, we should first use a more common substance that can have simple changes when the enzyme hydrolyzes it, such as color change. She did not know the exact answer to such a test but she promised to search for it.
A few days later, Prof. Li informed us that she found a special substance that can easily change color when exposed to β-glucuronidase, called Phenolphthalein β-D-glucuronide. If the enzyme has activity as expected, the substance will release phenolphthalein and show a pink to purple color when standard base solution is added.
Thanks to Prof. Li's help, we were able to establish a quick, easy and low-cost method for β-glucuronidase detection; and we changed our experiment plan accordingly.
A second interview with Prof. Li
We had the pleasure to invite Prof. Li over to our lab to offer some guidance on experimental details and experiment design. Prof. Li is experienced in cell culture and cell experiments, such as using the famous HeLa cell to study herbal medicine effects. In our lab, Prof. Li emphasized on the safety aspects and require all students to strictly follow the safety protocols, in the hope that one day some of our students could have a chance to operate on cells which required more complex protocols to avoid pollution and infections.
We reported our experiment results with Prof. Li on using Phenolphthalein β-D-glucuronide, which indeed was easy for us to determine if the expression of the enzyme was successful or not. Prof. Li suggested we could do at least one set of HPLC run to examine the enzyme activities and compare with the results in our lab. In addtion, Prof. Li suggested we could add a step to test the effect of glycyrrhetinic acid on HeLa cells to confirm the effect on cancer suppresion.
10. Lunch with UESTC
We consulted the UESTC team about the usage of Nanodrop and HPLC on Wechat. It was such a coincidence that our labs are close to each and we decided to have a lunch meeting. During the lunch we learned so much about HPLC and iGEM experience of college students, and the UESTC teams offered to share with us the HPLC technical manual they developed for their own experiments. Later in October, we received their little gift, which is quite heartwarming.
Stage 4: Project Communications
11. Interview with TCM Pharmacy
To let more people know about the synthetic biological methods for producing herbal extracts, we visited Guang-Yu-Yuan, a traditional Chinese medicine pharmacy with a history of over 400 years. Dr. Yang from Guang-Yu-Yuan kindly communicated with us and passionately learned about our project, and confirmed as pharmacist he will welcome the usage of our product. He also introduced to us the change underway in traditional Chinese medicine. From the introduction, we learned TCM is changing the way herbal medicine is raised and harvested.
Guang-Yu-Yuan Pharmacy for Chinese Medicine
Pharmacist prepares herbal medicine for brewing
Dr. Yang talked about safe drug usage for herbal medicine, which later can be found in our documentary
12. Interview with Tongfang Pharma
Tongfang Pharma uses modern science to analyze TCM effects and drug effects. In our visits, we introduced our project where we could use an enzyme to produce an effective ingredient in licorice. The specialist Dr. Xiaojia Chen from Tongfang Pharma was interested and suggested we consider broader application of our enzyme. Since the basic theory behind the project is that the enzyme can cut of glucuronide groups from glycyrrhizic acid to obtain our target, the glycyrrhetinic acid, would there be another substance that can be converted by the enzyme? Dr. Chen advised us on a few issues that we should look into, including the different activities the enzyme might have on different substances, the toxicity of the substances during lab research and production, how we should test and measure the production of those substances, and etc. Our project scope was opened up by her suggestions, and we integrated these considerations into our future plan and proposed implementations.
13. Interview with a veteranarian doctor
Our team captain Rachel got a chance to contact Dr. Liu from Lan-Qing-Yu Pharmacy. Dr. Liu specializes in the treatment of animal diseases with herbal extracts. Dr. Liu refused his appearance on a photo but warmly offer to chat with us over the messages. After our introduction of the project, Dr. Liu was excited to learn that using synthetic biology techniques the cost of GA, and similar herbal extract can be lowered because in daily usage, herbal extract are effective cures in many cases of animal disease. However the customers' requirement for an extremely low cost cure prevented the scale-up of applying herbal extract methods on animal disease.
References
[1]. Jones, J., Vernacchio, V., Lachance, D. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci Rep 5, 11301 (2015). https://doi.org/10.1038/srep11301
[2]. Adams, A. M., Kaplan, N. A., Wei, Z. et al. In vivo production of psilocybin in E. coli. Metabolic Engineering, vol 56 p111-119, (2019). https://doi.org/10.1016/j.ymben.2019.09.009.
[3]. Afkhami-Poostchi A., Mshreghi, M., Iranshahi M., et al. Use of a genetically engineered E. coli overexpressing β-glucuronidase accompanied by glycyrrhizic acid, a natural and anti-inflammatory agent, for directed treatment of colon carcinoma in a mouse model. International Journal of Pharmaceutics, Vol 579, 119159 (2020). https://doi.org/10.1016/j.ijpharm.2020.119159.
[4]. Wang, X., Feng, X., Lv, B., et al. Enhanced yeast surface display of β-glucuronidase using dual anchor motifs for high-temperature glycyrrhizin hydrolysis. AIChE Journal. Vol 65, e16629 (2019).
[5]. Wang, Z., Pei, J., Li, H., and Shao, W.
Expression, Characterization and Application of Thermostable β-glucuronidase from Thermotoga maritima. Chin J Biotech Vol 24(8), 1407-1412 (2008).
[6]. Wang, Guojing. Enzymatic Production of Glycyrrhetinic Acid and the Separation Process Design. Master Thesis, Beijing Institute of Technology. (2016)
[7]. Brittona, J., Majumdar, S., and Weiss, G. A.
Continuous Flow Biocatalysis.
Chem Soc Rev. Vol 47(15): 5891–5918. (2018) doi:10.1039/c7cs00906b