Team:Tsinghua/Human Practices
Integrated Human Practice
Questionnaire
Because our project is design to solve the environmental problem, our design should not only consider the experimental plan, but also the public's awareness of the issued problem and the acceptance of our product. In order to investigate the public's understanding of synthetic biology and biofilm pollution, as well as their opinions on our products, we designed a questionnaire for the public. Our questionnaires are produced, distributed and collected through Tencent Questionnaire Platform. Our questionnaire contains 16 questions, and we receive the results from 801 participants across 17 provinces. And this information has helped us improve our experimental plan and had a better design for the specific application scenarios of our product.
The first and second questions we designed include a brief introduction to Biofilm and our project, mainly aimed at helping participants to understand and promoting our No-mediated Biofilm Allayer project as well as what we are tackling with: the Biofilm.
Question01 About biofilm
Biofilm, which are bacteria packaged by materials secreted by itself, is formed by colonies of bacteria. Formation of biofilm improves bacteria’s resistance to antibiotics and disinfectant. In other words, it becomes harder to clean up. Biofilm is commonly seen in liquid pipeline environment.
Known Others
Question02 About our projects and products
iGEM team Tsinghua 2020 focus on use synthetic biology methods to solve the pipeline biological membrane pollution. We use Escherichia coli as engineered bacteria. The engineered E.coli is able to recognized the specific signal molecules, AHL, which is produced by biofilm, and produce nitric oxide (NO) through the nitric oxide synthase (NOS), so that the biofilm will be degraded, and therefore could be removed. And then, the engineered bacteria degrade as there is a light-dependent element in the engineered bacteria as well, in order to avoid environmental pollution.
Known Others
Question03 Your age?
12-17 18-24 25-29 30-39 Above 40
In the distribution of participants’ age, the youth (age 18-25) and the elder (age above 40) share almost the same proportion (42.6% and 46.9% respectively). While there is little subjects from other age groups. .Our questionnaire participants included young and middle-aged age groups indicating a certain credibility. And these age groups are most likely to be exposed to work dealing with biofilm contamination.
Question04 education background
Primary school Junior high school Senior high school undergraduate Master and above Have no formal education
According to our participants’ education background, “undergraduate and above” accounts for the highest proportion (84.2%), while the uneducated and who is not high on academic level accounts for little proportion (less than 0.5%). The participants shows a high level of education and ensures the credibility of their suggestions.
Question05 Have you ever seen this kind of “dirt” shown below in your daily life？
Yes. And I know its composition.
Yes. But I do not know its composition.
No.
Question06 The dirt showed above is actually biofilm. In your opinion, where might it appear? (choose more than one)
Drainage and water supply pipeline
The pool and the bathtub
Infusion tube in hospital, breathing machine tube, etc.
On dry table
Surfaces of surgical instruments
Tooth surface
In Question 5 and 6, we placed some pictures of biofilm pollution in common life scenarios such as pipelines to investigate whether biofilm pollution is common in daily life and to investigate the public's understanding of the nature of biofilm pollution. Most of the participants have seen the “dirt” caused by biofilm before (95.3%). However, most of them do not know it is biofilm that result in the dirt. This suggests that biofilms are very common, but the public may not have a deep understanding of their potential hazards like infectivity. This needs certain science popularization work.
Question07 Do you think biofilm would cause bad effect to our daily lives？
Yes No Senior Others
Question08 Do you think that biofilms may have adverse effects on daily life and production?
Clogging pipes
Affect appearance
Iatrogenic infection
Impact on food safety
Oral health problems
Others
Yes No Senior Others
Question08 Do you think that biofilms may have adverse effects on daily life and production?
Clogging pipes
Affect appearance
Iatrogenic infection
Impact on food safety
Oral health problems
Others
Questions 7 and 8, we hope to investigate whether the public knows about the hazards of biofilms. Basically, most participants can choose several specific hazards of biofilms. Almost all the participants considered biofilm having bad influences on our daily lives, which also suggests that our products may have some demands and applications in these scenarios.
Question09 In your opinion, which methods below could remove biofilm more efficiently and completely? (choose more than one)
Using brushes or dredging machine or other tools to remove biofilm artificially.
Using detergent, disinfectant or other reagents to remove biofilm chemically.
Others
Almost all the participants (90.8%) considered that chemical methods are accessible, while less than half (41.4%) participants considered physical methods working well too. In fact, chemical drugs and disinfectants are currently the main methods for dealing with biofilms and are also the most widely accepted.
Question10 Do you know about iatrogenic pollution in the hospital?
Yes Don't understand Others
In addition, P.aeruginosa is an important iatrogenic pathogen, which often causes iatrogenic infections in hospitals. Over 3 quarters (76.7%) of the participants do not know iatrogenic infection. We hope that people can understand the harm of iatrogenic infections, so that our products can have higher acceptance and more value for application.
Question11 Have you ever learned about Synthetic Biology?
Yes, but I don't know the details or definition.
I know the research content and definition of synthetic biology
Don't understand
other
Because our project is based on synthetic biology methods to solve environmental problems, we hope to improve the public's understanding of synthetic biology, so that they can more accept our product design. Participants who are merely heard of synthetic biology and who have completely no knowledge of synthetic biology take up the highest proportion (42.7% and 43.2% respectively). Only a little of participants (13.5%) can give the clear definition of synthetic biology. This reflects that the public does not know enough about synthetic biology, and we also need to do more HP projects and product promotion in the future, so that the public can more accept the application of synthetic biology in solving environmental problems.
Question12 A large number of studies have confirmed that the molecule of nitric oxide (NO), which is easy to be decomposed under normal conditions, can degrade biofilms, and a very small amount of NO concentration (mM-uM level) can make biofilms depolymerize and fall off. What do you think is feasible to use NO to clean biofilms in actual production and life? * choose more than one
The synthetic NO gas is directly injected
A synthetic NO chemical donor was used
Using engineered microorganisms that can produce NO continuously
I don't think any of them will work
other
Question13 If there is a biofilm cleaning product containing engineering bacteria harmless to human body, would you be willing to accept its application in daily life?
Questions 12 and 13 are aimed at investigating the public acceptance of our product for removing biofilms with engineered bacteria that produce NO. The majority held neutral and positive attitudes (grading above 5) towards using engineered bacteria to eliminate biofilm. Only 2.5% of the participants totally rejected this idea. Most of participants (70.7%) considered that the engineered bacteria which could generate NO constantly is accessible when dealing with biofilm. Less than half of the participants (40.1%) considered that chemical donor of NO also works. Only 8.1% of the participants considered that using NO is not accessible. With the average acceptance of 7.7/10, it shows that our product design can be accepted by the public.
Question14 What do you think is the potential safety hazard of using engineering bacteria that can produce nitric oxide (NO) to clean biofilms? * choose more than one
NO gas is slightly toxic
Engineered bacteria may cause secondary microbial contamination
Engineered bacteria may upset the ecological balance
No safety hazard or low safety hazard
other
Question15 What other comments and suggestions do you have for our project?
Questions 14 and 15 are designed to know the public's understanding of the potential safety problems of our products and collect suggestions for our products. This is one of the most important things to consider before our project products are applied. Over a half of the participants considered the secondary pollution, toxicity, and the destruction of ecological balance might be the possible bad effects cause by engineered bacteria (72.5%, 58.8%, 62.3% respectively). Less than 10 percent of the participants considered that engineered bacteria is completely safe. The majority think that our project needs to consider the potential environmental and ecological hazards of the engineered bacteria. This requires us to improve the design of security measures such as the kill-switch and to conduct further safety assessments in the future. At the same time, quite a few people believe that the toxicity of NO is also a safety issue. In the subsequent experiments, we further improved the detection of NO expression to ensure our NO level.
Field Interview of Chenming Swimming Stadium
Our team members are interviewing the engineers
The Chenming Swimming Stadium of Tsinghua University is a water sports stadium located in Tsinghua University in Beijing. It is also one of the water sports stadium for the 2008 Beijing Olympic Games and Paralympic Games and the 21st World University Games. The stadium has swimming area, diving area, dry diving training center and the physical training and recovery center inside. And the total pool volume is over 3500 m³, with average visit of more than a thousand people per day.
The swimming pool in Chenming Stadium and our team members are interviewing faculties
The water pump of the water processing system
We interviewed faculties of the stadium, including Mr. Feihong Lu and Mr. Sun, engineers in charge of water quality management and testing in the Stadium. They showed us around the spectacular water processing facilities under the stadium.
The interviewing group and Mr. Feihong Lu and Mr. Sun in front of Chenming Stadium
In addition, we introduce our knowledge about the biofilm and anti-biofilm project and they gave us their views on bacterial control in and biofilm contamination in swimming pool, along with precious feedback on our anti-biofilm project and the application of our product.
The team members are introducing our project
As they showed us, the water processing system of the swimming pool mainly includes water inlet pipe (into the swimming pool), water outlet pipe (out of the swimming pool), dilution tank, sand tank, disinfection tank, activated carbon tank and pump, etc. Before the Chenming stadium was renovated in 2017, ozone was the main disinfectant. Now, they're using a relatively harmless substance: sodium hypochlorite, as the main disinfectant. The PH of swimming pool is 7.85 and the total number of bacteria detected in the pool water was approximately 0. The free residual chlorine value was kept below 0.4 PPm there is no longer a strong smell of chlorine in the swimming pool.
The automatic hypochlorous acid control equipment
Through our interview with them, we know that the current methods to clean the biofilm in swimming pool, especially water supply pipelines, are lacking. As for the current facilities, the Chenming Stadium uses automatic control and detection equipment to make sure that the water inlet pipe will maintain a high concentration of hypochlorous acid for disinfection. So there will be little biofilm contamination in the water inlet pipe. However they think bacteria, of course, reproduce quite quickly. If the addition of disinfectant is stopped, the inside of the pipeline will become cloudy soon. Under the condition of maintaining a stable high concentration of disinfectant, it is difficult for the bacterial film to grow in the water inlet pipeline. But when hypochlorous acid is gradually diluted in the swimming pool, the biofilm may grow in the drainage system and the water outlet pipelines. The faculties think that there are not many directly effective ways to remove biofilm in the water outlet pipelines. The only way to clean them is to increase the circulation inside the pipe and increase the dosage of disinfectant. If biofilm is near the end of the pipeline, it can be cleaned by machine, but it is difficult to clean the inside of the pipe.
The engineers gave us recognition for our projects and ideas. But they also point out that if our engineered bacteria is not resistant to high concentration of chlorine, it may be of little effect in the water inlet pipelines. At the same time, they also suggested possible application scenarios for our project:
1. In most swimming pools that use ozone as the disinfectant, especially in those gyms. Because ozone is harmful to human body, it can only be added to the swimming pool in closed hours, and in the daytime, the swimming pool will not use ozone. One disadvantage is that there is no guarantee that a certain concentration of disinfectant will be maintained in both the pipe and the pool water all the time. So during the opening hours of the day, the lack of ozone provides an opportunity for bacteria and biofilm to grow in both the pool and the pipelines.
2. In the water outlet pipelines and drainage in swimming pools like Chenming Stadium. These are the places that lacks the high concentration of hypochlorous acid. Biofilm is easily grown in these environments.
3. In the sand tank and the dilution tank for filtration and biochemical reactions. The concentration of hypochlorous acid in these cylinders is lower than that of the water inlet pipe. These two tanks are mainly used to dilute hypochlorous acid and filter precipitation. The water flow in sand tanks and dilution tanks is also slower and may be contaminated by biofilm. Our engineered bacteria are also more likely to survive and play a role in degrading biofilm.
The water outlet pipelines and sodium hypochlorite disinfectant
Online Interview of Doctor in Peking Union Medical College Hospital
In order to investigate the application prospect of our products in the medical field, we contacted and interviewed Doctor Chai Wenzhao, director of the Infectious disease Department of Peking Union Medical College Hospital. Due to the epidemic management in and out of the school, our interview was carried out online, and a total of seven members in team Tsinghua participated in the online interview.
Dr. Chai Wenzhao, director of the Infectious disease Department of Peking Union Medical College Hospital, gives us much advice during the interview
According to our project, we hope to understand hospital prevention and treatment measures for iatrogenic infections, especially P.aeruginosa infection. Dr. Chai mentioned that P.aeruginosa is a very common kind of iatrogenic infection in hospitals, especially when disinfection techniques are not thorough enough. P. aeruginosa mainly lives in humid areas, so many hospital instruments, including surgical instruments and ventilators, can harbor the bacteria. To prevent iatrogenic infection, hospitals including the Union Hospital mainly focus on equipment disinfection and cleaning method. They have certain procedure and standard of disinfection and cleaning. In luminal instruments, including ventilators, bacterial species should be identified first by the infectious department in the event of widespread iatrogenic infection. The second step is to conduct drug resistance to determine the type and method of drug and disinfectant and have it disinfected by the equipment department. At present, hospitals have different sterilizing pools or cabinets and different sterilizing water for bacteria-contaminated instruments. Ventilator pipes have special high-pressure cleaning tools and ozone disinfector. Another important way to prevent iatrogenic infections is to use disposable equipment. In some hospitals, ventilator tubes are disposable and each patient can use one.
The online interview with Dr. Chai in Tencent Meeting
Seven team members of Tsinghua participate in the interview and had an in-depth discussion with Dr. Chai
Dr Chai was also positive about the application prospects of our project, but he also pointed out that if our project needs to be applied, we need to carry out an all-round assessment of the potential medical and environmental problems caused by our engineered bacteria. For example, if our engineered bacteria are not cleaned thoroughly, will they cause an immune response in humans. For now, getting the medical community to fully embrace the idea of “killing bacteria with bacteria” is a little bit difficult. But Dr.Chai suggested that we can start by cleaning the biofilms in medical instrument cleaning devices. The cleaning devices of medical instruments, if not adequately disinfected in the long-term cleaning process, will also be infected with many bacteria including P.aeruginosa.
During our interview with Dr.Chai, we had a better understanding of hospital measures to prevent iatrogenic infections. P.aeruginosa is a very important pathogen in the medical field. However, due to the high cleanliness requirement of the medical environment, there is still room for improvement in our project, especially the measures to ensure medical safety including the kill-switch design and safety assessment. Dr Chai also offers advice on the future application of our project.
Online Interview of professor Jin Fan
Prof. Jin Fan is the professor of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. His group mainly focus on the mechanism of bacterial biofilm formation, the gene circuit of Pseudomonas aeruginosa, an opportunistic pathogen, and the interaction between the bacteria and the host via interdisciplinary cross research.They also use optogenetics to realize the establishment of a common gene regulation circuit based on cycloguanosine diphosphate in the biological chassis of Pseudomonas . Their controllable biochassis and new genetic circuitry to try to solve major problems directly related to the environment and health.
After communicating with iGEM team SZU_China, they recommended us to contact with Prof.JinFan, who offered them many helps on their projects and perfectly matched our directions. On September 9th , we had a online interview with Prof. JinFan and had a deep conversation, involving the culturing of biofilm, quantification of biofilm and the feasibility of our design.
Prof. JinFan pointed out that the culture media is essential to the biofilm formation. Bacteria tend to aggregate and form biofilm under relatively harsh conditions, therefore medium with less nutrient, like M9 medium, instead of LB medium, is more appropriate for biofilm culturing. He also pointed out that quorum sensing system in bacteria is more complicated than we expected, and the process of biofilm formation involves the interaction of multiple QS molecule. This suggestion inspired us to detect the C4-HSL concentration in our laboratory cultured biofilm, in order to determine the feasibility of our proposal. We rethought that bacteria extracellular matrix might be a more stable and reliable choice for the switch than QS signal.
Offline communication with Prof.Liu Jintao
Prof. Liu JIntao is the professor of School of Medicine, Tsinghua University. Using interdisciplinary and quantitative research methods, his research group is committed to developing innovative techniques of bacterial membrane culture and observation, studying the population behavior and spatiotemporal dynamic characteristics of bacterial membrane, and looking for effective ways to treat bacterial infection.
Since we wanted to observe our biofilm formation and dispersion in flowing condition, we contacted Prof. Liu for the possibility of using micro-fluid chip for our dynamic experiment. On August 10th we went to Prof.Liu’s Lab and asked for his suggestions. Prof.Liu showed great enthusiasm on our project and offered us with much constructive advice, which layed the foundation of our hardware.
1.Conducting experiments on Microfluidic devices requires the design and build of the chips, while the processing period is about several months, so it is not recommended to use microfluidic devices due to the limited time.
2.As a substitution for micro-fluid chip, he suggested that the pipeline could be replaced by latex tubing or infusion tube. The culture medium could be stored in a simple container(eg: infusion bag) and continuously transported by peristaltic pump.
3.For a better observation of biofilm, it is recommended to transform plasmid with fluorescin into P.aeruginosa, and observe the dynamic of biofilm growing in certain transparent container using fluorescence microscope.
Throughout the period of preparing for iGEM, Prof.Liu’s Lab had provided many guidance and suggestions, involving the observation and quantification of biofilm. The strains WT P.aeruginosa and P.aeruginosa labeled with GFP were also rendered by Prof.Liu’s Lab. We are grateful for all the supports and instructions from Prof.Liu’s Lab.
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