Team:Shanghai High School/Human Practices

The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

Integrated Human Practices

Summary
Our research project was inspired by the notorious Changchun vaccine incident, which produced rabies vaccine using expired raw materials, leading to a large number of social dissatisfaction and human casualties in China. Considering the close correlation between our topic and social views, we conducted a large number of questionnaires and scientific popularization promotions. In such a series of activities, we were committed to spreading our topic to more people and gaining more social recognition.
Our dry group completed the design and production of our own branded products, and raised more than 1,500 RMB through our promotional and social media advertising. Our main way of fundraising was to release online fundraising activities through social accounts, and hold offline fundraising activities to attract more people to raise money for us by selling our customized products. In the offline fundraising activities, we raised a total of more than 2,100 RMB for later product research and development of offline science popularization. In fact, through these activities, we also carried out the project publicity, so that more people know our team.
We expanded our social influence by creating our official social accounts and publishing articles, which have been read more than 1000 times in total.
We also conducted a large number of offline questionnaires to collect data and information from the public, and by reading their opinions on vaccination we can improve the process of studying the RNA thermometer. At the same time, the questionnaire deepened their understanding of vaccine safety and helped us know better about public views on our program.
Next, in view of the practical application of our products, we conducted two interviews with experts. Once, we communicated with Dr.Hanbin Zhu about vaccine transportation and other issues online, and communicated with Dr.Yu Zhang about the practical application of our products in the laboratory. Through these two interviews, we determined the actual application of our products. We also listened to the advice of the general manager of a Chinese storage company to see if RNA thermometers could be used to monitor food safety. After visiting large food storage warehouses, we learned about the whole process of food transportation, including whether our products are suitable for food temperature testing. The wet group members were also given the opportunity to work with Dr. Li in the lab to test RNA thermometers. They used tryptophan, yeast extract, NaCl, water and AGAR for lysogenic growth.
Last but not least, considering for the social impact of our team, we made two community presentations to explain the importance of vaccines and vaccination to the community residents. In these two popularization of science activities, we got a better understanding of the public's attitude towards vaccines through the questions about vaccines raised by residents and their concerns about hyperthermia vaccines. Their attitude also further confirmed our determination to continue our research. And in these lectures, we explained some of the myths about vaccines through clear explanations.
The above activities that we conducted in the Human Practice section have effectively helped our group to improve its social influence, and we also collected useful suggestions for the experiments from professional professors. The trend of public opinion and public opinion highlighted in the questionnaire also guided us to solve the confusion in our research direction.

Purpose
-The expectation of RNA Thermometer:
To achieve our goal of designing the RNA Thermometer, we expected to find out several specific sequences of RNA with high sensitivity of detecting the surrounding temperature. By testing abundant trials of RNA sequences and the most suitable temperature of several different vaccines, we were looking forward to matching each vaccine with its favorable RNA Thermometer since each vaccine has their own favorite temperature. By doing so, we were able to ensure each vaccine has their own RNA Thermometer and to know if it would deteriorate during the transportation. Making sure the qualities of any vaccines is the fundamental purpose of our RNA Thermometer Design. Medical personnel could easily and accurately identify the safety of vaccines that were equipped with RNA Thermometer by checking the color changing on the thermometer. For our further expectation of this instrument, we would like to develop different color change to match their corresponding surrounding temperatures. In the future research, in order to transmit more precise information of vaccines, we are looking for one way that RNA Thermometer could express different color change after exposing to the environments with higher temperature.

According to the research we did on our research topic, we found some related study of RNA thermometer. Someone tried to use RNA thermometer to control bacterial heat shock and virulence genes.
According to their findings, we could know that RNA thermometer are located in the 5 '- underslated region of temperature - controlled mRNAs. From another research, the  modular nature of riboswitches and RNA thermometers were explored. The researchers illustrated that RNA thermometer can sense small molecules or changes in temperature. In this study, we confirmed our research direction, which is to conduct more accurate temperature detection through RNA thermometer.

 

Design
"Food shelf-life indicator"
Bread, milk and other fresh foods usually have a shelf life of only a few days. Once expired, these foods, which are easy to grow by microorganisms, may endanger people's health. The shelf life of these foods is closely related to storage temperature. For example, some food packages describe it as 3-4 days in summer and 5-7 days in other seasons.
However, this description of shelf life is not accurate. The shelf life of food shall be determined by the food producers and traders themselves according to raw and supplementary materials, production techniques, packaging forms, and storage conditions. Generally speaking, some kinds of food shelf life in the industry have been agreed into common warranty period, such as most of the instant noodles shelf life in 6 months; For milk, ultra-high temperature bactericidal milk is basically 6 ~ 9 months, pasteurized milk shelf life is 7 ~ 15 days, storage conditions are 2 ~ 6℃, but due to different packaging, production process, the final shelf life of milk is also different.
Here, we made a biological color display system closely related to temperature and time. The principle is to use cell-free translation - translation system (TXTL) to express pigment protein. Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. All of thefoundational knowledge from in vivo work on E. coli applies readily to E. coli cell-free systems. Although cell-free expression systems have applications in synthetic biology, to date the goal of most cell-free expression systems has been the maximization of protein and metabolite yield. People have developed an endogenous E. coli cell-free expression system that preserves the efficiency of protein expression demonstrated by previous systems but adds additional versatility by allowing expression and regulation based on both endogenous and exogenous (T7 or other) mechanisms.
Temperature-sensing RNA molecules, called ‘RNA thermometers’, operate in diverse natural contexts, mediating cellular behaviors such as heat-shock response and pathogenesis (9). There are many mechanisms for the operation of an RNA thermometer, each of which depends on a temperature-dependent conformational change. At its simplest, an RNA thermometer consists of an RNA sequence containing the ribosome binding site (RBS, Fig. 1A). At a permissive temperature, the ribosome can access this RBS and translation proceeds efficiently. At restrictive temperature, the RNA sequence folds in such a manner to prevent this access and inhibit translation. While those natural examples can have a relatively complicated secondary structure with multiple stems, hairpin loops, and bulges, RNA thermometers have been designed with simpler secondary structures, with only a single stem-loop protecting the RBS.
RNA thermometers, which are sensitive to temperature for a few hours after the liquid in the packet has fully reacted, can produce fluorescent proteins after being left at high temperatures for a few hours, allowing people to immediately notice potentially problematic foods. By screening RNA thermometers and reaction conditions controlled by different temperatures, as well as the temperature requirements of different foods at different temperatures, the shelf life of food can be evaluated according to the color indicator of food.
This project is of great significance to China's fresh food industry. Food safety is closely related to public health, especially fresh food in daily life, such as milk and bread. Fresh food has a short shelf life and is related to storage temperature, so it is difficult to track the freshness of each food. By attaching TXTL color indicator on the packaging shell, it is convenient to know the status of food. We are committed to developing a more accurate and sensitive biometric temperature recording indicator system, so that food can be safely delivered to consumers without spoilage.

Citation：Design of a Toolbox of RNA Thermometers Shaunak Sen, Divyansh Apurva, Rohit Satija, Dan Siegal, and Richard M. Murray
ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017
Downloaded from http://pubs.acs.org on April 27, 2017
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Zachary Z. Sun*1, Clarmyra A. Hayes*2, Jonghyeon Shin3, Filippo Caschera4, Richard M. Murray2, Vincent Noireaux4
URL: https://www.jove.com/video/50762 DOI: doi:10.3791/50762

Our original goal is to design the RNA thermometer to detect the safety of food. After a few days of experiments and a period of group discussion, we decided to expand the range of our research topic to test the temperature of vaccine to guarantee its validity. We expect to find an expert to explain several relevant questions. Thus, Dr. Hanbin Zhu is the best choice. Dr. Zhu is a former president of Wuhan Jianghan District Red Cross Hospital and the director of Medical Department, Wuhan Institute of Technology Hospital. We had an interview with him. In this interview, we learned a lot about our topic-RNA thermometer and other related knowledge, like how to transfer vaccines, how to test the vaccines. Refrigerator and refrigerated truck are the most widely used methods of transportation that can maintain the temperature at 2 to 8 degrees Celsius to guarantee the effectiveness of the vaccines. He also said a vaccine management law will be enforced in December. After that, the safety and quality of vaccines will be improved because of the stricter law. Moreover, he believes that there will be a better, more effective and more accurate method to check whether the vaccines can be used or not. For our topic, Dr. Zhu confirmed the practicability and validity of our research. According to Dr.Zhu’s suggestions, we obtained more academic knowledge that is helpful to improve our experiments and ensure that the topic can be completed.
It was our pleasure to invite Dr. Yu Zhang, a professor of Chinese Academy of Sciences. He assured us of the value and market’s acceptability of our product – RNA thermometer to test the validity of vaccine. First of all, He reminded us that the  vaccine preservation standardsare very strict. Even though vaccines didn’t necessarily need to be stored in a sterile environment because vaccines were already sealed, temperature must be strictly controlled within a range. In fact, most vaccines consisted of proteins or other organisms’ macromolecules. These macromolecules are likely to break down under room temperature or high temperature. Dr. Zhang also gave us some advice if our RNA thermometer cannot express clearly: increasing the number of plasmids for color expressing, rearranging the sequence of RNA for transcription and translation, and increasing the brightness for florescent proteins. Furthermore, Dr. Zhang encouraged our product to enter the market when it is complete because he suggested that RNA thermometer would be functional if the cost could be controlled; however, our product’s target users are limited within a few categories, such as hospitals and labs. Dr. Zhang believed that if our product became a necessity in one profession, then the product could be granted with success.

Survey
Online
In the past decade, emerged a series of inferior vaccine events. Depending on the news, the event was born with weak supervision. As the years gone, governments pay more and more attention to the regulation of the security of the qualified vaccine. Under this situation, they may meet with high pressure and high density of work. To relieve the working pressure, we are just trying to invent a kind of the RNA thermometer which can change color remarkably to show the quality of the vaccine. It turned to make it easier to determine whether vaccines qualify or non-qualified.
Collecting public thoughts is a necessary way to enhance our study. Thus, we created a survey covering a wide spectrum of our topic. To bring together the ideas efficiently, both online and offline questionnaires made. Moreover, the questions in the survey are intended to be multiple choices and a definite choice to improve the effectiveness of the survey. There are definite differences between the paper offline questionnaire and the online questionnaire. The online questionnaire is aimed at enhancing people's understanding of the overall situation of vaccines at this stage, while the offline questionnaire is slightly more focused on the development of our experimental products. The purpose of the questionnaire is to determine the ideas and opinions of the public and then to have a clear idea and direction for the project. We first published an online questionnaire. As for offline, we send leaflets to passers-by on August 6th.
From the result of the online survey, we learned that people are quite curious about the quality of vaccine since 44.12% of the interviewees responded positively to this issue while 23.53% of the interviewees expressed concerns about vaccine producers. About 77% of the interviewee suggested that they can remember the adverse reaction after use of the vaccine as well. Also, people are more inclined to employ the production circulation link quality inspection to protect the rights and interests of citizens. Hence, the staff can easily verify the quality of the vaccine. If the assumption (vaccine thermometer), the step will be highly optimized for distinguishing inferior goods. Hence, we found the necessity to develop our product.
Do you have any adverse reactions after vaccination？

What do you think are the main sources of vaccine safety risks？

Now there is a defect in the temperature indicator of the vaccine. If there is a new design that can solve this problem, would you like to support this project？

On the 9th of August, we, Dr. Thermometer members went to Putuo district and did a popularization for the elders. This activity mainly has three goals: To enhance the elders’ understanding of vaccines and how to deal with invalid vaccines; to give them a more precise picture of vaccines; to promote our products.

It is clear that the majority of the elders have somehow cared about the question that is conquering the world – are vaccines safe?

According he pie chart above, 45% of the people that we surveyed paid more attention on the safety of vaccines. Among the surveyed people, those who cared most about the date of production and effective duration of vaccines were at the same percentage – 21%. When receiving vaccines, only a few people regarded the company that produced vaccines as the most important factor they should care about.

Based on the data concluded from the survey, there are people caring about all four sectors in the duration of the whole process of the vaccines. Compared to the percentages of the rest, percentage for people caring about the production process of vaccines was higher.

Last but not the least is to ask the elders to fill in the questionnaire, which would help us to have a more complete understanding of their views on vaccines.
According to the questionnaire, we can tell that over 90% of the elders or their family have the experience of getting vaccines. Although some of them are not sure about if they have been vaccinated or not. It is obvious that none of them have not been vaccinated.

Also, 82% of them are concerned about problematic vaccines and the vast majority are worried about qualities of vaccines when they are vaccinated.

50% of them always trust Community vaccine sites, 33% of them would choose to go to National inspection and quarantine bureau and 17% still count on hospitals.
As we predicted before, most of them cares more about the safety of vaccines, also the sate of manufacture.
We do not see any geocentric in the data of this questionnaire, 40% of them think the domestic vaccine is more suitable for their body.


The great majority of people will not trust a vaccine when they do not have background information of the vaccine, however, they are positive about the safety of domestic vaccine levels.


On August 12, 2019, we went out into the community and gave a presentation to a group of elders, whose average age is above 60. We explained a basic understanding of the vaccine itself, introduced the safety of vaccines to them, and indicated the comparisons of local and imported vaccines. Moreover, we introduced them our products in a simplified way. At the end, we conducted a survey among our audiences to enhance the current citizen’s’ understanding of vaccines. This allows us to ensure the impacts our products will bring to the society.

Execution
Today, we came to Shuyun (Shanghai) supply chain company to look for the possibility of our product investment in fresh food storage warehouse. Stepping into the normal temperature warehouse, we know from the manager about the warehouse operation process: get the order, check the order to pick up finally to the loading; each step is assisted by various information systems. For example, OMS system is responsible for receiving orders for the warehouse; WMS system is responsible for helping to allocate the goods to be loaded; TMS is responsible for making the order to the driver. Then he took us to visit the storage room of frozen materials. The management mechanism is very meticulous, and all food and drinks are regularly arranged in different warehouses. During the interview with the warehouse manager, we know that the warehouse has a clear label corresponding to the temperature layer from the entry end. Every time the new products are put on the shelves, the detailed information (picture, brand and storage conditions) will be added to the system for maintenance. When the products are put on the shelves, the warehouse will automatically remind us which warehouse the materials should enter. In this case they can facilitate the multi-temperature layer management, avoid errors. Finally, we go to the quality control manager to interview Mr. Wang, the engineer. He said the factory has had few problems with material distribution and storage and food testing.

Q:What the sampling method dose the company apply?
A: If the number of commodity is less than 10, every commodity will be checked. If the number of commodity is more than 10, at least 3 percent of the whole commodity will be checked. If we check out any problem of commodity, the inspection ratio will increase.
Q:Can you tell us how your company has implemented multi-temperature management to store food?
A：There are clear corresponding temperature layer labels. Every time new products are put on the shelves, we will add detailed information (pictures, brands, storage conditions) to the system for maintenance, and the system will automatically prompt the materials to enter warehouses.
Q:Is there a power outage in the warehouse? What is your company's emergency response after the power outage?
A:It would first ensure that the workers in the factory were evacuated to the outside. After that, it was sent to check whether there was a problem with the electric line. If there was a problem not because of the factory itself, it would use the extra generator to produce electricity.
Through this activity, we realize that dramatic improvement should be made to put our products in the fresh food industryThe systematic operation mechanism in Shuyun inspired us to enhance the rigorous design of our product for future application in fresh food storage.. At the same time, thanks a lot for all staff’s cooperation!

Offline
Meanwhile, the offline survey gives us a similar suggestion. Over 86% of respondents show their confidence in the government's regulations. However, there are still 7% of people doubt about them. Thus, the problems of ensuring vaccine safety still exist. Still, 47 people (41%, which is the most of all) said that the vaccine problems were caused by the weak oversight by the government. It seems that a good indicator applied in the supervision of transporting vaccine is still important. The result again strengthened our confidence in developing products. Originally, we thought that offline research activities were well designed and implemented, but in practice, we learned a lot of lessons. First of all, when we were looking for someone to fill out the questionnaire, we couldn't even analyze the best location for the respondent. After a several trials, we decided to choose the leisure area like KFC, McDonald's, and Starbucks to conduct our survey since people there ususally have time. Meanwhile, when we send the questionnaire, we should first introduce the purpose of our questionnaire, so as not to let the other party think that it is a pyramid scheme. Finally, we divided our work and sent out questionnaires one to one. In this case, the efficiency can be greatly improved. We gained a lot in this event and provided a lot of experience for our next scheduled study. At last, the survey solidifies our ambition in inventing the RNA thermometer. Nearly 100% percent of respondents suggest that they are willing to support the idea of using heat shock proteins to visualize vaccine status. They all agree with the idea that the vaccine indicators are needed to be fixed. We will continually develop our experiment progress.

 

Education & Public Engagement
Official Account
In order to introduce our team to the society and raised social awareness in our community, we established an official account on WeChat. Based on our team name, Dr. Thermometer, we thought of a similar yet straightforward name for our official account, Thermo Lab.
In our published articles every other day, we included information such as the safety of vaccines and the danger of receiving invalid vaccines, as well as updating our progress of this project. The first article in the account provided samples of products designed with our logo, to receive support from our subscribers in order to obtain enough funding for future advocacy. In addition, we attached a concise description of our team and objective. Then, we "confessed" the progress happened in the next few days: we visited a local supply chain Inc. and met up with a few professors in the field of genetic sciences.
With citizen's enthusiastic support, we had successfully allow more and more people to understand our topic and this iGEM competition, and become aware of the validity of vaccines when receiving. In addition, we had approximately 1,000 views on our articles. At the end, we made a total of 28 sales of our items, and the fund acquired from which were used to cover our expenses in consulting experts to ensure the value of our product.

 

Lectures in community 1
Background：
On the afternoon of August 9th, we went to GanQuan community in PuTuo district of Shanghai to conduct scientific popularization of our products and vaccine-related knowledge. In the early stage of the activity, we carried out a large number of popular science content，activity promotion and other aspects. Finally, we got in touch with the community leader and launched such an activity.

Procedure：
In the promotion part of the activity, we designed a poster to introduce our activity, the iGEM competition and the whole team for publicity and Posting in the community. To attract the audience, we also prepared snacks and napkins as gifts. Our main promotion content is to carry out science popularization and publicity. We told the audience about our RNA thermometer, the iGEM contest, and the whole team. At the same time, we also explained the relevant knowledge of the vaccine, including the meaning of the vaccine, the over-temperature vaccine, the reasons for vaccination and so on. In order to keep the content from being boring, we designed a delicate PPT and selected interesting video. After the explanation, we distributed questionnaires to the audience to supplement the data of our offline questionnaire survey and make it more comprehensive.

Feedback：
Through a slides presentation and video demonstration, we introduced to the audience the varieties of vaccines, the importance of vaccination and the emergency measures after vaccination. Since most of the audiences are old people, we increase the trust and familiarity with each other through the use of dialects and simplified professional vocabulary, and break down every complex professional knowledge into easy to understand content. At the end of the activity, they also had a full understanding of what we have popularized, and showed great expectations and support for our products. They also said they would introduce our team to their children.

Conclusion：
Through such an opportunity to communicate with the elderly in close proximity, we promoted our products to more people and groups, and publicized the whole iGEM competition, gaining greater social recognition. Secondly, we take advantage of this opportunity to explain some inherent wrong ideas , fully achieving the purpose of popular science. The event ended successfully and raised social awareness for our team.

 

Lectures in community 2
Background:
When we approached the end of our designed project, we realized that the understanding of our product was absolutely necessary in our society because we needed to contribute a high communication with people who actually related to the product after all. Meanwhile, we made our posters, several slides and even some videos that directly related to the idea of our project to introduce our design to public community. By doing so, introducing our project to them was not the only purpose, but making sure the qualities of the vaccines that they probably would use in the future became more critical.

Procedure:
We customized around 100 questionnaires according to the number of citizens that we probably would meet. For the investigation, we designed 12 questions in each questionnaire in order to investigate people’s concern about the safety of vaccines. We also brought some souvenirs to the people who did the questionnaire.

In the morning, we did presentations about vaccines and the product that we designed in the laboratory. Our videos also contained several general ideas about the situation of vaccines in people’s daily life. After the introduction, we distributed our questionnaires and helped them to finish each question in order to make sure we know the public concern about the vaccines. In addition, we gave our souvenirs attached with our QR code to encourage them to visit our official website so that people who had better ideas could communicate with us immediately.

Feedback:
After we distributed our questionnaires and QR codes, we quickly increased the views and subscriptions which means more and more people payed their attentions to the safety of vaccines. After analysis of the data that we collected, we found that 86.7% people highly concerned the safety of the vaccine and 66% people payed a lot of attention on producing and conserving the vaccines. The question data showed that people already changed their perspectives on vaccines, which means both government and scientific teams should give enough concern and protection to the vaccine project. Some people also highlighted that the transportation of the vaccine would influence the quality of each vaccine if there was no protection during the transportation.

 

Lectures in offline fundraising
Background:
In the summer of 2019 on August 11th, in order to better for offline popular science of presentations, Dr. Thermometer team held a large-scale offline fundraising activities. We invited many teachers, friends and parents of the team members to this fundraising event.

Procedure:
Before the activity began, we made a propaganda. We designed two posters, one to fully show our fundraising activities, and the other to publicize our popular science project. We also distributed fans with our promotional content in shopping malls, subway stations and educational institutions before the event to attract crowds. We typed our logo and activity information on the fans, and we distributed 500 fans totally!  Our activity was held in the busiest activity area in central Shanghai, and the audience was mostly teachers and students who were interested in our research. We started with an introduction to our team and the iGEM competition, followed by an introduction to vaccines and the importance of vaccination. After that, in order to liven up the atmosphere, we held three simple and interesting games, such as Hangman. After everyone started to actively participate in our activities, we tried to attract the crowd with performances. Rin and other students performed singing and interesting dubbing. After these activities, we began to appeal to all the audience to help us with our research projects by purchasing products designed by us, such as hats, clothes, key rings, etc.

Feedback:
Thanks to your kind help and hard work, we have sold more than 30 pieces of products, among which the key chain has the largest sales volume. Finally, the total revenue is 1269.9 yuan. Many parents have followed our official social media accounts to follow the progress of our experiment. This part of funds we have used for offline publicity.

Conclusion:
It was a success because we managed to get a large number of people to know about our program and vaccine related expertise. At the same time, we have successfully completed the sales without any loss. The core of this activity is to help our project go more smoothly, obtain social funds and open our social influence. We have achieved these goals and raised funds for the later stage of the project, which ensures that our activities can be carried out in the later stage.

 

©2019 Microsoft Corporation. All rights reserved.