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How to communicate with different stakeholders
How to solve problems in project development
How to improve the project from different perspectives
How to consider ethical issues

In order to allow human practice activities to play a greater role in the project, our project team members have carried out a variety of unique activities around different aspects of the project at different stages, and provided constructive suggestions for our project development so that we can Be able to know the role and influence of our project from different angles. Throughout the process, we paid special attention to epidemic prevention and the privacy of interviewees, and we obtained many unexpected gains.

How to communicate with different stakeholders

Public

Our team wants to evaluate the public acceptance for portable test kits and their personal requirements. Considering the danger of the COVID19, we decided to conduct survey in the form of online questionnaires in order to prevent team members and the public from being infected by the epidemic.
A total of 647 replies, covering 16 provinces of China, have been collected. Based on cross-analysis of the data under the objective factors such as age, education, and residence of the questionnaire, we obtained plenty of useful data for the project.

Part of the analyzed data is shown in Figure 1.

Figure 1. Data analysis of the survey

Overall, the public acceptance for portable virus detection device is relatively high. 32.6% of people said that they are willing to try the instrument if it is accurate, which is in line with our initial design. Meanwhile, 11.5% of people said that they would be tested by the people with professional medical or health knowledge instead of home testing, indicating that it is auxiliary to set up a virus detection device in a small medical institution.
Moreover, 7.9% of people said they would try the device if it is harmless to the body and our device exactly meets the requirement. Only 3.3% of people refuse to use home testing kit, as they think the results will not be so accurate. Thus, the portable virus detection device is acceptable by the public.

"Which of the following viruses do you prefer to be detected by the home test kit"data analysis chart

Table 1 Needs for virus detection

People have diverse needs for home testing and the detecting virus types, which confirms that the multi-virus detection device meets the market demand.

Medical institution

At the beginning of the project, we want to put the portable detection devices at clinics or community hospitals, hoping the surrounding residents can be detected by the professionals, which can also be used in remote areas.
However, we gradually realized that our thoughts were not feasible after we visited the clinics and community hospitals.
According to the doctors, only second-level and above medical institutions are qualified for virus testing in China. However, clinics and community hospitals are all first-level medical institutions, which will not own the certificate for virus testing.
Based on the investigation results, the project planning has been strictly adjusted, aiming to simplify the device operation and transfer our project products to the second-level and above medical institutions.
In order to reduce the trouble of medical staff, we contacted them in advance before the communication, followed their opinions and did not take any pictures during the entire survey activity.

The company

To further know the market demand of the virus detection device and solve some technical problems in the project, team members and the instructor went to Bioer Technology Company for investigation. We presented our projects to the company and conducted in-depth interviews with the technical director, marketing manager and general manager in the company.
(Please click the link to go to the module of Bioer Technology in Science communication )

Help to Dry Lab:

The technical director in Bioer suggested that we add a pair of filters at the incoming light and the outgoing light to get a cleaner filter in order to reduce the influence of LED scattering, which was taken into account in the design of our fourth-generation device. In addition, Bioer Technology sponsored us with four sets of expensive filters (Figure 2).

Figure 2 Filters provided by Bioer Technology

Help to Wet Lab:

When designing the fluorescence readout experiment, we were afraid that crRNA and synthetic target RNA in the detection system would be degraded by RNase from the air, leading to the failure of the experiment. Some helpful advice were gaven by the GM of Bioer. First, all of the reagents and experimental equipment need to be treated by DEPC water and the bench surface need to be cleaned by RNase Away reagents to make a RNase-free environment. Second, don't keep focusing on the RNase and consider other reasons if the experiment failed or no positive result was obtained as the influence of RNase in the environment would not be so significant based on her previous work experience.
In the subsequent tests, the experiment was not so successful at first. According to the GM's suggestion, we consider other reasons rather than struggle with the RNase. Eventually, we were lucky to get a positive result.
For the practical application of the project, they reminded us that protectants and desiccant should be added into the protein during freeze-drying. Otherwise, the protein would get damp quickly in the environment, resulting the failure of preservation. For lyophilization, they suggested us to consult Cepheid company. We have included the company's investigation in the Implementation plan to optimize the practical application module of the project. however, we did not complete this work due to the limited time.

Figure 3 Investigation in Bioer

Assistance to the Human Practices:

1. On the issue of the market environment for portable virus detection devices raised by us, the general manager and marketing manager of BIOER said that the market for portable virus detection devices is very promising and that many biotechnology companies are currently developing them.
2. For the final marketing of our devices, we also asked about the marketing process for such medical testing devices in China. The general manager patiently explained the following steps to us:
① Conducting market research to determine the feasibility.
② Determining the method by the think tank.
③ Registering related products.
④ Trial production(Clinical work in at least three hospitals)
⑤ To the Chinese Academy of Forensic Identification.
⑥ Clinical comparison: compared with existing methods, the accuracy rate is required to be above 90%.
⑦ Submitting appraisal reports and clinical reports.
⑧ The expert group shall conduct assessment to determine whether the company is qualified for producing, testing and obtaining the registration certificate.
⑨ Producing and marketing.
⑩ The whole process takes two to three years, even three to five years.
3.We would like to know how to dispose of the resulting medical waste. They said that medical waste is recycled by specialized enterprises. If there are non-communicable wastes, we can contact the EPA to develop a recycling programe.

How to solve problems in project development

The long-term communication with GenScript

We divide the content of the exchange into two modules.

Probe design

After half a month of communications and discussions with GenScript and over 20 emails, we were able to take into account the fluorophore structure, synthesis path and other issues with the assistance of the company. After four major revisions, we finally completed an important element of the project-the design of fluorescent probes. Moreover, GenScript also instructed us to conceive a control trial of cutting preference with or without thiomodification.

The details of communication with Genscript:

This part of the exchange with GenScript greatly promoted the component design of our project, and also brought our project one step closer to realization.

Detection experiments interview questions

We encountered difficulties in fluorescence readout and data analysis in the detection experiment. We communicated with GenScript’s PhD in primer design on these issues. For the four probes we chose, because the excitation and emission wavelengths corresponding to the fluorescent dyes are within the same range, we do not know how to choose the excitation and emission wavelengths required for actual detection. For quantitative analysis, we should draw a standard curve of the probe connected to RNA but not connected to the quencher to see the maximum fluorescence value that can be achieved and the detection limit of the microplate reader under this condition. The initial experimental design did not Consider this. We redesigned our experimental program according to the advice of GenScript experts, so that we can complete the qualitative testing and Part characterization before the deadline.

Cooperation with QHFZ

We confirmed a long-term partnership with QHFZ, after a period of communication in various aspects. The lyophilization of Tardigrade intrinsically disordered proteins (TDPs) they studied has solved the problem of decreased protein activity in the lyophilization of our Cas. The technology of our project has been improved theoretically to make our project more perfect.

Interview the professors

We tried to seek help from teachers to promote the stagnant project process.

Dr.Sun:

　　　　In Part construction of the project, we accidentally made a mistake which led to the wrong part while adding His-tag and SUMO tag to CcaCas13b gene. Our initial idea was to try to remove the redundant HIS-tag by one-step PCR cloning, while Dr. Sun proposed that the HIS-tag could be removed by PCR directly through changing PCR primers. Finally, we succeeded in removing the redundant HIS-tags according to Dr. Sun's suggestion. Another question is whether redundant His-tag will affect protein activity if the extra His-tag label was not removed. Dr. Sun insisted in removing the extra His-tag label . He also raised questions in the assaying of the recombinant protein expressed from the Part. He proposed that we should perform the subsequent purification and digest after in vivo activity confirmation.

Dr.Ge, a professor in the field of molecular probes

At the mid-stage of the experiment, limited by the laboratory apparatus, we had a problem that the precision of the enzyme-labeled instrument is not enough to obtain the best emission wavelength when measuring the lower concentration probe and using the theoretically excited wavelength scanning emission.
To this, we visited Dr. Ge in the field of molecular probes and she put forward that fluorescence signal can be amplified by changing gain value. Also she reminded us that when comparing experimental data of different boards, we need to control the same value of gain, and Rt-pcr instruments were recommended to try to use. The second problem may be that the concentration is too low and the excitation emission is too close together. It is suggested that the reaction probe should not be used first, but the dye should be used to explore the conditions first.
The teacher's suggestion helped us to overcome the limitation of laboratory instruments, so that we could better deal with the experimental results.

Dr.Liu, an expert in chemical engineering

For the unique device unit, we encountered difficulties in the process of bold innovation exploration. As to the prevention of bubbles occurence in the device, Dr. Liu gave us suggestion that due to the capillary phenomenon , no bubbles will be generated and samples can be wetted when the diameter of the channel is less than 10 mm according to our device design. This helped us a lot to overcome the difficulties of device design.

How to improve the project from different perspectives

Communication to promote the overall development of the project-CCiC

The four-day CCiC conference is a rare opportunity to learn synthetic biology and its related fields. In order to expand all aspects of the project and communicate with more teams, we joined CCiC. After the meeting, we received high-level feedbacks from the CCiC Committee, as well as souvenirs, pointing out the issues that needed to be addressed urgently in our project.
We have made some adjustments to the project based on the suggestions:

1. The model still needs in-depth improvement, and the selection of parameters is still problematic. To establish a model that matches the experiment, we must work hard on the parameters, including consulting the database and referring to the literature. We must not fit the parameters with quite low under the condition of very low experimental quantity. The contingency is extremely unlikely and of universal significance.
2. The core of the model is Michaelis-Menten equation. It is not recommended to use the same empirical equation in large quantities. Some parameters may not be consistent.
3. The model of multi-virus detection system also needs to be considered, the direct expression of different fluorescent proteins affects each other, and the inhibition of substrate on products can be considered.
4. The demonstration works well; the detection principle seems to target RNA viruses.
5. Pre: The video is quite impressive, with plot and scene design, but the transition and promotion of various parts of the content is somewhat abrupt. For projects, it is very good to use a variety of combinations of the more mature tools of Cas 13 cutting, and try to make a materialized test strip. Attention should be paid to the biosafety assessment of E. coli expression mimicking viral protein capsid-coated RNA, as well as to the effect of coating. The part of fluorescence detection needs to pay attention to the threshold range of confirmation and judgment of positive and negative. Modeling is still relatively simple. Cas 13 cutting dynamics modeling is a bit rough, it is recommended to refer to other Cas 13 cutting dynamics parameters.
6. Poster: At present, the poster is quite good. The main color is obvious, the sections are clear, and the arrangement is more appropriate.

Cooperation to improve project integrity-virus database and WeChat applet construction

In order to further enhance the integrity of the project, we want to realize the interaction between the user and the test results. We propose to store the basic information of a variety of viruses in the form of WeChat applet, which is convenient for users to query viruses and obtain relevant suggestions. In order to facilitate professionals to query the virus crRNA information, we also want to build a virus database to store the crRNA information of various viruses.
After early contact, we decided to cooperate with the team of Shanghai Jiaotong University. We introduced our idea of building virus database and functional requirements, and provided them with virus database data and page layout style. After listening to our sharing, they put forward some corresponding suggestions on how to build the database, and they are willing to help us on this issue. In addition, they also shared with us their experience in making applets, which is of great help to our applets building. With the efforts of both sides, we have completed the basic functions of the virus library.
Virus database related code, our partner SJTU has been put into GitHub. If you want to know more, please click >>Viralibrary<<

How to consider ethical issues

Personal safety of the team

Due to the impact of the 2019-nCoV, we intend to avoid contact with the outside world as much as possible and give full play to the Internet. We have carried out activities such as education for children, questionnaire research and online meetings.
All the activities are planned in advance. If there is any activity requiring field research, we will take temperature measurements, wear mask and alcohol-based hand sanitizer and other safety measures.

Physical and psychological safety of the interviewees

We have great respect for the rights of the interviewees. Before the visit, we communicated by phone and email and obtained the consent of field research. When visiting, we take pictures only with the permission of visitors and follow their rules and regulations. After the interview, we sorted out the research results and put them on our Wiki with the consent of the interviewees. It is for this reason that we have not included a frontal image of minors in the education.
During the investigation of the biotechnology company, our mobile phones were labeled with anti-shooting labels. Our feet were covered with shoe covers, and we wore masks. All through the investigation, we followed the guidance of the person in charge. During the clinic investigation, we followed the respondents' wishes and did not take any photos. For the online survey questionnaire, we set the interface very succinct. In order to protect the privacy of interviewees, they can directly fill in the questionnaire without user authentication, and users can choose to quit the questionnaire interface during the interview.

Team:ZJUT China B/Integrated human practice

Integrated human practices