Team:NUDT CHINA/HP Overview
HP OVERVIEW
After identifying the project, we continued to conduct extensive literature research while conducting public-facing pre-research. We hope to get the following results from this study:
(1) Understanding the public's understanding of synthetic biology and iGEM provides reference for our popularization science day activities, and science lectures in ChangJun High School and Southern Medical University. Besides, accurately locates the public's cognitive level and points of interest, and realizes that science and technology are truly public-oriented and close to society.
(2) Through this research, we hope to obtain an evaluation of our team's project and demonstrate the significance of our project to synthetic biology and society.
We collected 952 valid questionnaires and obtained the following results:
1.1 Public Concern
The high public interest in biosafety and medical costs is fed back into the experiment, reminding our team to focus on biosafety and research and development costs in the design of the pilot line and in the development of late-stage experiments.
Figure 1. Pie chart showing public concern.
1.2 Public Acceptance
Figure 2. Pie chart showing the acceptance level towards synthetic biology of public.
1.3 Potential user needs
In the questionnaire settings, we statistically analyze the potential user's needs for the project application, and conclude that there are the following:
Figure 3. Bar chart indicating the need of potential users of our synthetic biology project.
We analyzed and visualized the results of this public-facing questionnaire survey, and obtained the above suggestions and evaluations which have been fed back into the work of the experimental group and the popular science education activities in the later stages of HP. This is a pilot survey conducted by the project, which demonstrates that a practical requirement for protein degradation and controlling the cycle of mammal cells in various fields. Furthermore, it reminds us of the considerations in the experimental design, links the laboratory with the society, and provides the argument and support for the project.
2.1 Interview of Prof. Chun Meng
Brief Introduction of Chun Meng: Professor of the College of Biological Science and Engineering of Fuzhou University. His main research direction is biopharmaceutical and tumor diagnosis. He also has done some research on the properties of single chain fragment variable(ScFv).
The Interview Process:
When we has already found the sequence information of scFv targeting cyclin E, we started the experimental design. In the meantime, basing on the collaboration reached with Fuzhou University, we contacted Professor Chun Meng, working as the professor in Fuzhou University and having a certain research on scFv, for an interview. We introduced our design and hoped that he could give some comments or suggestions on our design.
Professor Meng highly commented our work and said it was "very enlightening". At the same time, he also gave some potential problems of our design. He pointed out that the affinity between VH and VK of scFv is not so robust, and our split design may not achieve the ideal effect. In addition, since the sequence we found has already been published, its affinity to the target protein remains to be argued. Therefore, he suggested that we could conduct further research and find more types of targeting sequences to achieve the project objectives.
Figure 4. Screenshot of interview with Prof.Chun Meng.
What we learnt:
1. The split design may not achieve the ideal effect, so testing the binding efficiency is necessary.
2. It is necessary to consider the specific affinity of the targeting module and investigate more targeting sequences.
2.2 Interview of PhD Student Mirta Viviani
Brief Introduction of Mirta Viviani: She is a phD student, working on laboratory for Biosystems Engineering, School of Life Sciences, Westlake University.
The interview process:
When we completed the design of the blue light mediated interaction modules and introduced them into our Predator system, we were surprised to find the poor performance of degrading target protein, whereas the blue light interaction module has already been tested independently, where CRY2-CIB1 showing high binding efficiency under blue light illumination. With these doubts, we contacted Miss. Mirta from West Lake University hoping she could give us some guidance.
Miss. Mirta is very interested in our research on trim21 over the past four years. She comprehended the work we have done, then she analyzed some possible problems of the system from the perspective of protein structure, such as the proportion of interaction modules, the length of flexible peptides, the number of targeted sequences and so on. This inspired us to improve the length of flexible peptide in the system eventually, so as to obtain more obvious experimental results.
Figure 5. Screenshot of interview with PhD Student Mirta Viviani.
What we learnt:
It is necessary to improve the proportion of structural elements and the length of connecting flexible peptides of each part of the predictor in order to achieve better degradation effect.
2.3 Interview of Prof. Guodong Yang
Brief Introduction of Guodong Yang: Professor of the Department of Biochemistry and Molecular Biology of the AFMU, Ph.D., PhD supervisor, has done optogenetic research and cell cycle research. The main research directions are biology; basic medicine; cardiovascular system diseases.
The interview process:
We have a discussion with Professor Yang regarding the light-inducible protein pairs at the beginning of our project. We introduce our goal this year, and he show high interest of our work. He think our project last year is with high quality, and we can do a lot of work basing on it. He suggest us to search on the online databases to select protein dimerization pairs appropriate in our system.
Figure 6. Prof. Guodong Yang.
What we learnt:
1. The biggest advantage of light control is controllability. It is necessary to make full use of this advantage to find application outlets.The professor has studied the release of insulin. The release of insulin particularly requires rhythm, which is related to rhythm in terms of light control (We introduce that to CSU after we know that they are researching the release of insulin too).
2. Use online databases (like https://www.optobase.org/) to identify potential optoswitches that could function as core module (interface: in IGEM 2020 NUDT_CHINA) to generate a set of light controlled protein degradation tools.
2.4 Interview of Prof. Kou Zhenzhen
Brief Introduction of Kou Zhenzhen: Major in medical education and marginal subjects of medicine; endocrine glands and systemic diseases; military medicine and health; optogenetics.
The interview process:
In the interview with Professor Bian Huijie, she mentioned the possibility of applying our project in neurobiology, so with her help, we contacted Professor Kou Zhenzhen in neurobiology on September 1st.
Figure 7. Discussion with Prof. Kou Zhenzhen.
What we learnt:
In the field of neurobiology, the Predator system based on nanotechnology can be used for brain implantation to regulate the application of human body and cell rhythm. In the field of optogenetics, it is similar to the optogenetic project it is doing. It is similar to optical fiber. For animal experiments, the lower end of the cannula can be fixed to a certain brain area of the mouse by intubation method to target and degrade a certain protein to see behavior Variety.
2.5 Interview of Prof. Ru Yi
Brief Introduction of Ru Yi: Research fields: biology; surgery; oncology; neurology.
The interview process:
After interviewing Professor Kou Zhenzhen, Professor Kou recommended Professor Ruyi to answer our questions about targeted protein degradation and abnormal cell proliferation. Ruyi is also an expert in the field of neurobiology. Therefore, on September 2nd, we made a special trip to interview Professor Ruyi.
Figure 8. Discussion with Prof. Ru Yi.
What we learnt:
Targeted protein degradation. I have paid attention to the ubiquitin proteasome system before, and I see it mentioned in your project profile. The advice I gave the last two days is from the known and clear mediation of cyclin ubiquitination. Look for the binding sequence in the ligase E3 that is degradable by light. This sequence is generally not too large and can be loaded on your light-controlled on\off original to realize the degradation of cyclin controlled by light.
Abnormal cell proliferation is a characteristic of tumors. If it falls into reality, it must be confirmed that cyclin is expressed differently in diseased and non-disease tissues, and it is a very important initiating factor. Cyclin is regulated by multiple signaling pathways. In a practical sense, preventing upstream signal transmission may be more effective than inducing its degradation.
2.6 Interview of Prof. Huang Wan
Brief Introduction of Huang Wan: Research fields: chemistry; organic chemical engineering; medical education and medical marginal subjects.
The interview process:
During our interview with Professor Bian, Professor Bian introduced us to the possible applications of our project in cancer treatment. After the interview, Professor Bian affirmed our idea of further exploration in this direction, and recommended Professor Huang Wan to us to provide us with help in the next step. So on September 10th, we conducted an interview with Professor Huang Wan.
What we learnt:
It is recommended to focus on the application of the project on auxiliary treatment and rehabilitation.
In the case of tumors, the best way to find tumors is to remove them. If your project is to be applied to tumor treatment, intratumoral injection can at best prevent metastasis and other auxiliary effects. The better application direction is for postoperative rehabilitation, Or preoperative preparation, tumor detection, etc., for tumor cure, more reliance on resection, radiotherapy and chemotherapy. Your project is actually similar to chemotherapy. If it can be made into a “chemotherapy with less side effects”, it will be very promising.
2.7 Interview of Prof. Chen Yatong
Brief Introduction of Huang Wan: Research fields: skin diseases and venereal diseases; biology; medical education and medical marginal subjects.
The interview process:
As a college represented by engineering, we are still exploring how to embody engineering thinking in our projects, while combining the concept of synthetic biology to create modular and structured product applications. For this reason, we found Professor Chen Yatong from the Department of Cell and Molecule, AFMU, and interviewed her on September 18th.
What we learnt:
Look for more application outlets for extracorporeal therapy, such as designing engineering circuits, logical languages, and turning our projects into a "building block".
From the perspective of protect technology, the most important thing to degrade it is to keep this thing as small as possible. How your project penetrates into the cell is a relatively big problem. So in fact, the application scenario of your system is not possible in the body. It’s too big, so you’d better go outside and think of a way to make it something like a "building block"; or if you turn it into a circuit, I control this thing to the final output module, such as You become a logical language by activating a certain mode.
3.1 Interview of Prof. Huijie Bian
Brief Introduction of Huijie Bian: Deputy director of the Cell Engineering Research Center/Cell Biology Department of the AMMU, professor, Ph.D., doctoral supervisor, and visiting researcher of the German Cancer Research Center.The main research direction is tumor cell biology: targeted gene therapy, carcinogenesis and development.
The Interview Process:
After we established the topic and carried out the preliminary experiment, we wanted to know in what aspects our results could link with the society, provide beneficial value to the society, and solve some problems in the experiment. Therefore, we contacted Professor Bian Huijie and came to the National Key Laboratory of Macromolecular Cells of AMMU on August 28th, 2021 to ask her some questions about experiments and applications.
After we introduced the project, Professor Bian gave an overall evaluation of our project: "At the moment for the cell cycle inhibitors to target has not been very successful developed, to suppress the side effects of larger, cell cycle targets choice is always the key point of the drug companies, such as p53 tumor suppressor gene targets, at present although there has been a certain research results such as MDM2 regulation of p53 protein degradation, but overall still under exploration. You guys are coming down to the cell cycle, you're starting at E3, and I think your project has a lot of application and social significance. I hope you can further carry out your experiments and bring your results to basic medical research, and I believe you can make something meaningful for society."
In terms of experiments, for the problems of transfection expression, Professor Bian suggested that we design a control experiment and try to delete Trim21 in cells. Specifically, we can use CRISPR/Cas9 technology to knock Trim21 out or RNAi interference, and then import it into the Predator system. In the construction of stable cell lines, Professor Bian suggested that we construct sequences into lentiviruses to achieve stable expression.
In terms of application export, Bian said that the idea is feasible, combining nanotechnology and implanting in the brain to regulate certain cell cycle circadian rhythms. At the same time for inflammatory diseases such as psoriasis, it can be made into a similar form such as plaster transdermal medication, and then try blue light irradiation, specific dosage forms and other problems can consult some pharmaceutical companies pharmacist. In terms of genetic technology, if the targeted introduction of viruses is selected, such as Newcastle disease virus in oncolytic virus, it can theoretically achieve tumor therapy combined with blue light by taking advantage of its characteristics that it only replicates in tumor cells and not in other cells (may combine but not replicate). Intratumoral injection for tumors easily exposed to blue light, such as head and neck cancer, breast cancer, etc. Liver cancer can be interventional through intubation, such as tumor blood supply and other aspects. In general, local administration is theoretically possible for any tumor.
At the end of the interview, Professor Bian generously gave us some suggestions for the improvement of our system. She reaffirmed the significance of our project to the medical system and society, and was willing to help us in the subsequent experiment and promotion.
Figure 9. Interview Prof. Huijie Bian.
What we learnt:
1. We have learned about the cell line construction method of infecting cells with lentivirus, and at the same time solved the problem of transfection and expression in the experiment, which is helpful for the further development of our experiment.
2. We understand the application possibilities of the project in nanotechnology, skin disease treatment, cancer treatment, genetic technology, etc., which will help us to further explore how the project can build a bridge with society and can truly produce beneficial effects to society Influence.
3.2 Interview of Prof. Zhen Li
Brief Introduction of Prof. Zhen Li: Deputy Director of the Department of Histology and Embryology of the AFMU, professor, PhD, and doctoral supervisor. The main research direction is histology and embryology, endocrine system, reproductive system physiopathology research.
The interview process:
After we interviewed Professor Bian Huijie, we contacted Professor Li Zhen with experimental and application questions. On September 1st, 2021, we came to the Key Laboratory of Histology and Embryology of AFMU to interview Professor Li Zhen.
In the interview, with regard to the transfection expression problems we encountered in the experiment, Professor Li, like Professor Bian, suggested that we design multiple sets of controlled trials. In the construction of stable cell lines, Professor Li suggested that we should not emphasize transfection technology, because all kinds of transfection and expression technologies are becoming mature, and only highlight the highlights.
In terms of application export, when the professor listened to us to explain the project, the first reaction was the application to Alzheimer's disease, the degradation of β-amyloid protein (Aβ42). Professor Li made the following assumptions for us: "Can a nanodevice be made into the brain to specifically and accurately degrade amyloid β? Or can it be achieved through a similar method of minimally invasive surgery to intervene in the degradation of amyloid β? This is theoretically very feasible.” At the same time, Professor Li mentioned that when the pathology is caused by hyperproliferation and other diseases, such as myeloproliferative disease (MPD), intramedullary injections are performed to edit hematopoietic stem cells. In theory, It is also feasible.
During the interview, Professor Li also made some very important suggestions for the presentation of our project. The professor suggested that we reduce the emphasis on the heritage of the project, just as an "introduction." The professor said: "From my point of view, can you tell us about the work you did in the early stage when you perform, but you may not need to tell the detailed steps. For the findings of previous deficiencies, this is your research. The process, you don’t need to say it, you should focus on the advantages of this year’s results. My suggestion is: take the results of previous years as plan A, B, etc., this year’s project is plan C, and compare this year’s projects in parallel It is the optimal solution. I feel that this can improve the competitiveness of your project."
Figure 10. Interview Prof. Zhen Li.
What we learnt:
1. We have clarified the next experimental idea of designing a controlled experiment, which pointed out the direction for the further development of our experiment.
2. We understand the application possibilities of the project in Alzheimer's disease, myeloproliferative disease (MPD), cancer treatment, etc. This further broadens the application direction of our project and makes us more confident to carry our results Entering the hospital and entering the society reflects the significance of our research.
3. We got the idea of a project display, reducing the introduction of the early stage and focusing on the advantages of this year’s results, which is very meaningful for us to write and display the Wiki later.