Team:ZJU-China/Human Practices
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OVERVIEW
• Investigation about genetic engineering
During the Techfest, we did a profound investigation concerning about genetic engineering and public perference between cell-containing diagnosis system and cell-free system.With the devolpment of China, since reform and open, the past forty years has witnessed great improvement of people’s living standard. However,people’s knowledge and ideologies about genetic engineerng develops not as fast as the growth of economy.
Therefore, we designed a detailed and comprehensive investigation about public perception about synthetic biology, genetic engineering and people’s perference between cell-containing diagnosis system and cell-free system. Surveys includes basic knowledge such as “Do you think hybrid rice is transgene plants?” and detailed questionnaire about “what’s your main worry about genetically-modified organism and biological products?”This questionnaire give us some important consideration and adjustment about our project.
Questions about basic biological knowledge help us distinguish the target population possessing basic biological knowledge.
The high ratio of correct answer shows their knowledge is convenient for our investigation and their opinions are also relatively referable for us. And then we choose our target population to continue our questionnaires.
The deficiency of concept about Synthetic Biology shows importance and necessity of the Techfest, one of our education and public engagement activities. Therefore, we followed up with a question concerns about the cell-containing system and the cell-free system.
Confusions and worries are expressed through this question. Here, we see the advantage of cell-free system when biological products meet the public concerns.
To confirm the result of our last question, we keep on asking the public for their preference of a new diagnostic device. So, here again, the advantage of the cell-free diagnostic device shows up. Hence, to get a more detailed feedback from this public engagement, we designed 3 more questions in order to understand what is the exact worrying for public.
Results from this survey can be included as 2 points:
1. Public concerns are in line with mainstream opinions in China which is very conservative. We realized it would be a wise choice for us to develop a cell-free diagnostic device.
2. There still exists concerns in our diagnostic device from the public perspective even though it is already cell-free. Then, the problem turns to a safety-related concern which requires us to ensure there exists no cell left on our device.
Lianjun Lin, Director of the IVDs department of MDST.
Is our diagnostic device biosafe? Is it good for our community? Does it meet the demands of public? Can it be truly applied to our truly life? To complete the investigation of the big background where our ideas were born, we visited experts in related area and performed questionnaires in order to evaluate the public acceptance of our device.
A Public investigation concerning about the public acceptance of our device:
• Biosecurity & Ethics Consideration
Biosecurity is the most important and a basic consideration of our product since we chose to develop an in-vitro medical device. MDST (Zhejiang Institute of Medical Device Testing) is a supervisor department of government responsible for regulating medical device. Hence, we thought the suggestions from this department would make a great difference to our final product. Here, we applied a filed visit to their lab and interview the department charge, Lianjun Lin to consult his advice and opinions about the biosecurity and our medical device. We summarize his points of view which do make great impact on our project designing and list them below:
• Questionaries
Q: How to promise the security of a medical diagnostic device?
A: It’s hard to define the word “security” while it related to many other concepts. The first thing you should concern about you device security is the national standard of in-vitro medical device. It represents the minimum level of safety that a product can be allowed to produce. Particularly for your device, I recommended you to focus on the basic security concerning about the Cell-Free Biosensor.
Q: How long can a medical device be reached out and scalable produced? How can we be more quickly to transform technology?
A: It depends. Usually, the process of acquiring the permission of scalable producing takes 2-3 years, including the product research period, which is according to my working experiences. But this situation is for biotech company, not for a scientific research and innovation team like you. To transform your ideas, I recommend you to think about cooperating with biotech company involving with in-vitro diagnostic device. Additionally, you should take it seriously to protect your patent right while you are sharing your ideas to others.
Q: What are your suggestions in ethics while we are designing our device?
A: Ethics consideration is as important as the security of a medical device. Nowadays, the mode of Internet plus Hospital is increasing with the concerning on the privacy disclosure. So, my suggestion here is data security while you have a further development in your device which combines with big data and internet.
Q: What do you think of our product if it can be worked out? Can you picture out your ideal in-vitro diagnostic device?
A: I think what you’re doing is great which directs me to a different way to develop in-vitro diagnostic device. If you ask me the ideal IVD from my perspective, I would love to tell you that I want to have everyone have their family physical examination at home. I think your design will possibly give out a solution for this since it is able to use types of enzymes to detect different indicators. Keep doing!
Meet with CCiCTime: 2018.8.27-2018.8.31
Place: ShanghaiTech University
• Overview
As an 8-year-old team, ZJU-iGEM absolutely won’t miss this biggest iGEM conference in China! This year, CCiC attracted more than 80 teams to share their projects and exchanged their valuable experiences.
• Details
Also, we presented our project and drew much attention.Then,during panel session, Xuan Wang, an experienced iGEMer, post-doctor in Tsinghua University, told us that especially for the New Application Track, if we want to stand out, we must convince others by showing them a workable device. Since we were trying to construct a user-defined sensor, we considered to write a universal and detailed instruction which made it possible for everyone to construct his or her own sensor.
This year, we found that there were 2 teams trying to construct cell-free systems. And we exchanged our experience and shared our ideas on how to make a more distinctive ‘cell-free’ system. We all agreed that there is not a clear definition of cell-free. So, we further explored how to construct CFPS (cell-free protein synthesis) in a better way.
What’s more, our inspirations burst out when we communicated with other iGEM teams. How to predict our protein docking model was one of the biggest barriers toward our project’s completion, but after talking to Peking-iGEM, we surmised that their theoretical modeling can help us out! We were also worried about lacking negative control in Curli-Fibers Expression. Fortunately, UTSC-iGEM gave us a suggestion that we can use Acetobacter xylinus to express Curli-Fibers without SpyTag so that we could set it as our negative control when demonstrating the specific binding for our reconstructed Curli-Fibers.
In iGEM HQ face-to-face session, we sit around Mrs Meagan and discussed how to make iGEM projects better and lead iGEM competition to a more influential competition. We shared suggestions one by one. Also, through this session, we understood the significance of after-iGEM, and we knew how to break a complex project into parts and accomplish them one by one. We could take just several parts with breakthroughs to Boston for the competition and finish rest parts during following years. It also helped our team to inherit team culture.In iGEM HQ face-to-face session, we sat around Mrs. Meagan and discussed how to make iGEM projects better and lead iGEM competition to a more influential competition. We shared ideas with each other. Also, during this session, we understood the significance of after-iGEM, and we knew how to break down a complex project into parts and accomplish them one by one. In short, CCiC gave us a vast platform to communicate and explore ideas. Thanks, CCiC!
Expert interview• Uartar Seker, Assistant Professor, Synthetic Biology Group, UNAW
To enable our curli fiber a better conductivity, we enhanced it with nanogold particles. But we were confused on how to confirm the best concentration and the best size of nanogold particles for our experiments. Fortunately, we got in touch with Professor Uartar. He suggested us using 5nm gold nanoparticles to decorate our electrode so that we can maximumly fill up the gaps between curli fibers.
• Chao Zhong, Ph.D., Assistant Professor, Integrative Bio-inspired Molecular Engineering (IBME) Group, ShanghaiTech University
Our project aims to detect the current at the downstream of our enzymes’ reactions. But at that time, the problem was that the current was so weak that we could not detect them in a convenient way which definitely didn’t reach our expectations. So, after we searched papers on how to enhance Curli-Fibers’ conductivity, we turned to Professor Timothy and Professor Zhong for advice.
Professor Zhong advised us to fill up all the gaps above our electrodes. He said it would be much better if we filled up with nanogold particles and he also offered us his E.coli strain (PIN4) whose endogenous CsgA was knocked off. And finally, to help us catch up with more ideas, he suggested us to take a glance at his newest paper[2].
• Christopher Whitford, ZJU-China & Julius-Maximilians Universität Würzburg Mentor in After iGEM
Mr. Chris and ZJU-iGEM were firstly connected by After iGEM. He is an experienced iGEMer and such an earnest man. We had our first meetup in 8/8/2018, during which Mr. Chris pointed out that iGEM concerns not only the main body of your project but also the demonstration of the significance of your project. iGEMer should realize that it would never be useless to show your comprehensive considerations of your project’s Human Practice.
In the second meetup, we were confused by how to ensure the biosafety of our cell-free biosensor. He provided us his recent paper talking about how to apply the biosecurity of iGEM projects.
The third meetup was mainly talking about the Human Practice Part of iGEM project. “The more Human Practice for your project, the more competitive you project is.” We realized it would be a promising Human Practice activity for us to keep frequent communication with Acon-Biotech and have more cooperation with Sir Run Run Shaw Hospital.
We sincerely thanks for his selfless denote!
Integrated Human Practices Acon-Biotech• Overview
A field visit to in vitro diagnostic company, Acon biotech and a face-to-face talk with in vitro diagnosis industry veteran. To have a further understanding on in vitro diagnosis(IVD), we choose Acon-Biotech to perform our integrated human practice. Here, we got our main feedbacks and in turn, developed a mode of transformation of Scientific and Technological Achievements.
• Start from the field visit
ACON Biotech (Hangzhou) Co., Ltd. is a US living Chinese established foreign-owned biotechnology company, was founded in 1995, and is the one by China GMP, European CE, ISO13485 international certification creature diagnostics company.
Covering in vitro diagnostic reagents, medical equipment, medical electronics three major industries, high-tech products and services to customer-centric, integrated biotechnology (BT) and electronic information technology (IT), ACON has developed a large amount of rapid, accurate and convenient clinical testing products to facilitate the diagnosis of disease, and provide users with more convenient and abundant bedside diagnostic products. Now the ACON is one of the National Torch Plan Key High-tech enterprises and member of Hangzhou enterprise technological innovation and development association.
The research and development Manager, Mrs. Juan Cui, together with her colleagues had a table meeting with us. Actually, we didn’t aware where our cell-free biosensor’s advantage lies at the very first. But after a detailed discussion, they showed much more interests in our project. They pointed out that the most intricate problems in commercial IVD is the exorbitant cost, temporally and economically.
Below are the detailed reasons:
1. In their actual working process, firstly they have to test out the best enzymes matching ratios. As the enzymes are in a free state, the cluster reactions may not process easily and adequately, which makes this preliminary experiment much more complicated and highly-costed. This especially highlights the demand of our enzymes complex.
2. After getting the result of the best enzymes ratio, they will use a cluster of oxidase enzymes to detect the target substrates, and similar to the glucometer, the final result will be represented through the current transferred by the oxidase reaction. But the point is, this cluster of enzymes can only be single-used, which apparently, increase the cost of diagnosis.
What our user-defined in vitro diagnostic device can do:
1. It would be more efficiently to research how to find more in one detection. This is where our design originated – enzyme logical gates. Such gates can inform users of changes in multiple physiological indicators and make judgments about whether they are sick. By contrast, traditional detection methods require different sensors to detect different substances in sample, which requires a lot of precious time. We believe that the parallel processing capability of logic gates fits well with the high time requirements of Point of Care Testing, and it is expected to explore new possibilities in the field of first aid.
2. How to make our enzymes recycled? That’s a little bit hard but also a little excited to answer. The Curli Fiber, was first recognized as the invasion tool for bacteria, which firstly reconstructed by Harvard-iGEM 2014 as artificial biofilm, may help us out. This year, we use curli fibers with enhanced nanogold particles to covalently fix our enzyme complex on our electrode, which theoretically makes our electrode able to be recycle. And since it can be recycled, we can probably change the traditional detective methods or even lead a revolutionary among IVD.
• Keep in touch and continue our cooperation!
Above is the very first communication between ZJU-China and Acon-Biotech. Later, through frequent conversations, we even get more detailed information.
We summarize these feedbacks into serval points of view and list the corresponded adjustments to our projects.
Part I.
“Time matters. Time matters to personal experience. Time matters to human life.”
Frequent communications with Mrs. Juan Cui made us realize that the time required for detection determines whether our products are practical. To develop our product and focus on the concrete perspectives, we mainly focus on the optimization of the product response time. Especially in first aid, a one-minute delay can lead to irreversible consequences. After many rounds of brainstorming, we realized that instead of sticking to shorten the time required for a single test, we can splice our enzymes complex in-vitro with the help of Spycatcher-Spytag systems (there are other 2 orthogonal catcher-tag systems). And theoretically, this enzyme complex system can shorten the distances between each enzyme, which means this form of enzyme complex can couple cluster of enzymes’ reactions. This enzyme complex is relatively stable so that it can be applied on in vitro diagnosis.
Part II.
“Technology Transferring lies shortcut for nobody but for the explorers and man of perseverance.”
We are not content to rest on the results of the iGEM competition. We want more. Here, we summarized a developed and suitable mode for iGEM teams to build a strong connection with companies. Additionally, we are attending to transformation our project into practice and registered the part of “After iGEM”.
#the pdf passage# Sir Run Run Shaw Hospital• Project Design
Making something that can quickly detect indicators had always been our direction. Since it is characterized by rapid detection which we are going to utilize and find a suitable situation for, we turned to hospitals-first aid needs quick response! We went to SIR RUN RUN SHAW HOSPITAL, the third affiliated hospital of Zhejiang University for valued opinions of clinicians.
It is widely acknowledged that General Surgery is well-informed involved in emergency departments and a great many consultations, so we contacted the director of General Surgery who had extensive experience in this field. After learning our appeals, the director recommended several suitable applications for A Detector: poisoning, flu, myocardial infarction and first-aid. Symptoms of the diseases above are all not visible but requires urgent detection. Through WeChat and offline meetings, we discussed the feasibility of each proposal with him.
First, poisoning is relatively unpopular, and the poisons are diverse, which puts higher demands on high specificity and sensitivity of the approach to detect. The cost of translating research results into clinical practice is also relatively high and requires substantial funding support.
Actually, we are interested in studying flu because there are always countless people suffering in the spring and autumn in Hangzhou. If it can be detected by portable chips, it will achieve effective early-prevention, reduce infection and benefit the society, especially the elderly and children. However, God did not grant what one prayed for sometimes. Our campus lacked a corresponding laboratory with negative pressure to study virus and the headquarters of iGEM had strict restrictions on safety as well.
Therefore, we used to struggle between myocardial infarction and first-aid. If the time window of myocardial infarction is not caught, the patient would pay the price of disability and even death. So is first aid. Meanwhile, both the myocardial infarction and emergency biochemistry are also complicated and have urgent need for rapid detection. Whereas, as for which indicators are most suitable for our project, the director of General Surgery said that he could not give authoritative advice, for the director of Clinical Laboratory is the expert who he suggested we should go to.
Clinical Laboratory of SIR RUN RUN SHAW HOSPITAL has played an irreplaceable role in providing advice to clarify potential indicators. Although there were some indicators to choose, from our perspectives, we would like to do some general but practical work, the framework of which could provide reference for disease diagnosis and later research. After understanding our vision, he explained the basic items of CX7+ in detail and thought we could deal with basic indicators first within the scope of our current capabilities. Eventually, after several discussions late at night, we decided to use glucose, lactic acid and lactate dehydrogenase as signal inputs of detection.
During this process of project design, the directors highly appreciated our rigorous scientific attitude and had great expectations for A Detector.
• Modeling
The dry lab selected the classical clustering algorithm KNN in machine learning to help us classify samples as pathology or normal. The core assumption of KNN algorithm is that if the majority of the k most neighboring samples in a feature space belong to a certain category, the sample also belongs to this category.
We imminently need hundreds of patient data and controls to implement this model and then we turned to the director of Clinical Laboratory in SIR RUN RUN SHAW HOSPITAL again. He generously offered clinical data for modeling and blood samples for further product testing!
Here is the cooperation agreement signed by Clinical Laboratory of SIR RUN RUN SHAW HOSPITAL and ZJU-China. It said during iGEM Competition Clinical Laboratory would provide a certain amount of blood samples and related data to ZJU-China and were also responsible for safety training before clinical experiments. Accordingly, ZJU-China should protect the information of the data and the privacy of samples from infringement. All the materials obtained from Clinical Laboratory is for scientific research purposes only.
It was a bright sunny morning, but we had to come to hospital for data. We worked on the hospital’s internal data system from 9 a.m. to 7 p.m. and collected the data of more than 600 patients and controls after 2 days’ work.
The results of modeling indicate that the clustering model performed well within the scope of our acceptance when applied to unknown samples, which drives the progress of A Detector and makes the diagnosis more credible. Here are results of modeling.
Moreover, in our communication with other teams online, we found that the acquisition of data is pretty difficult, especially hundreds of large volume data, so we are pretty grateful to Clinical Laboratory of SIR RUN RUN SHAW HOSPITAL for the selfless help.
• Applied Design
In October, after rounds of testing using simulated samples in the laboratory of Zhejiang University, we took the products to SIR RUN RUN SHAW HOSPITAL for real sample testing. It is also approved by the experimental center of Zhejiang University.
At the very beginning, Doctor Jin of Clinical Laboratory gave us a comprehensive training on how to use a blood sample in a standardized manner.
We were supposed to wash our hands in six steps, then wear overalls, disposable medical masks and gloves.
In October, after rounds of testing using simulated samples in the laboratory of Zhejiang University, we took the products to SIR RUN RUN SHAW HOSPITAL for real sample testing. It is also approved by the experimental center of Zhejiang University.
We were designated to use the blood samples in blood collection tubes with a purple test tube cap. Doctor Jin introduced that the blood collection tubes contain EDTA-K2 anticoagulant, which is usually sprayed, so that the anticoagulant is evenly distributed on the wall of the tube, and the specimen is prepared for whole blood, which is generally suitable for routine hematological examination.
1. Put a blood collection tube into the Allegra X-12 Centrifuge series of universal desktop centrifuge, centrifuge at 3000 RPM for 5min, the supernatant is the serum needed.
2. Unplug the purple test tube cap and use a disposable dropper to draw a certain amount of supernatant (serum) from the tube to the surface of the modified interdigital electrodes.
3. Immediately cover the purple test tube cap.
4. Throw the used disposable dropper into the waste bin specially for biohazard.
When we have completed the preparation and protection, the following is the specific compliance protocol (under the guidance of Doctor. Jin).
5. Collect the electrode-washed liquid containing serum to the waste bin specially for liquid biohazard.
Under the supervision of Doctor Jin, we performed several repeated exercises using real blood samples and then started independent experiments on the prototype of A Detector.
The whole process of product testing was full of twists and turns, but we eventually got experimental data of applied design! Here is our applied design and the video.
Generally, we learned the importance and influence of A Detector to the world in the following aspects through communication with clinicians.
If A Detector can be popularized, the physiological indicators of the patient can be measured in time and avoid missing treatment time window. Thus, the survival of critically ill patients will increase to some extent.
Since A Detector can be considered as a platform and the demands of hospitals are diverse, various customized detectors will be born. For long-term goals, if A Detector are intended to be used in public, it will face more stringent requirements in more aspects. Meanwhile, as we concerned about the practical application for A Detector, we consider this human practice a precious chance to have a further understanding from the perspectives of biosecurity and ethics that related to patents’ using experience. And this definitely extends our works in the transformation of scientific and technological achievements.
There is no denying that A Detector still have a long way to go!
References[1] Barnhart, Michelle M; Chapman, Matthew R (2006). "Curli Biogenesis and Function". Annual Review of Microbiology. 60: 131-47. doi:10.1146/annurev.micro.60.080805.142106. PMC 2838481. PMID 16704339
[2] Wang J, Polsky R, Xu D (2001). "Silver-Enhanced Colloidal Gold Electrochemical Stripping Detection of DNA Hybridization". Langmuir. 17 (19): 5739. doi:10.1021/la011002f.
[3] Wang J, Xu D, Polsky R (April 2002). "Magnetically-induced solid-state electrochemical detection of DNA hybridization". Journal of the American Chemical Society. 124 (16): 4208-9. doi:10.1021/ja0255709. PMID 11960439.
[4] Daniel MC, Astruc D (January 2004). "Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology". Chemical Reviews. 104 (1): 293-346. doi:10.1021/cr030698. PMID 14719978.
[5] Hu M, Chen J, Li ZY, Au L, Hartland GV, Li X, Marquez M, Xia Y (November 2006). "Gold nanostructures: engineering their plasmonic properties for biomedical applications". Chemical Society Reviews. 35 (11): 1084-94. doi:10.1039/b517615h. PMID 17057837.
[6] Heitner-Wirguin, C. (1996). "Recent advances in perfluorinated ionomer membranes: structure, properties and applications". Journal of Membrane Science. 120: 1-33. doi:10.1016/0376-7388(96)00155-X.
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