Team:NKU China/HP/Gold

In our Human Practice project, we decided to do a survey focusing on the health and safety of researchers. We collected information and the results inspired us a lot. We consequently decided to integrate the 'safety' part into our program.
We will demonstrate the survey before presenting more details of the integration.
Context, rationale & prior work of the investigation
Event 1 On Sep 21st, a serious accident hurt three students badly, which shocked the whole country. According to the report, a student who is in his second year of postgraduate study was teaching two newcomers to do experiments. He wrongly used an Erlenmeyer flask instead of a beaker and added much potassium permanganate (KMnO4) to concentrated sulfuric acid. When he was doing this, he told his students to be careful because "the product is easily to explode". Pitifully, hardly when he had finished the sentence did it come true. Two of the students became blind with their face destroyed.
Event 2 Young Overseas High-level Talents Introduction Plan is a program started by the government of China. The program encourages young scientists to return and work in China. With the help of the government, talented young scientists can do research freely. However, many young scientists work so hard that they don't care about their health. They work for so long time that many of them suffer from serious health issues. According to a survey made by China Association for Science and Technology, scientists work for 8.6 hours per day in average, the longest of which spend 16 hours a day working in lab. Things are particularly worse for PhD students. They work for 9.29 hours per day. Recently, a professor died at the age of 35, the best age for scientists.
Traditionally, respect hard-working people are highly regarded. This tradition influenced all the East Asian countries. In Japan, you could still see that many people work as long as they could. Another reason is that the government used to encourage scientists to devote themselves to their work. This is because China used to be poor and weak in 1950s. Many scientists devoted themselves to the country and scientific research, which was badly needed at that time.
But now China is a confident and powerful country. As Chinese scientists are doing better and better, they shouldn't be encouraged to work for too long simply in order to publish more papers. They should be encouraged to care about their own health, instead.
Rationale: We hope to change the life of Chinese young scientists and we wish them to be safe and healthy. The most important part is to change their working habits. We decided to start from master and PhD students because it's a crucial period for them to form their habits of working, which might influence their whole career. The more people we could influence, the larger changes there will be.
Information collecting
What are the most common poisonous reagents in biology laboratories? We have asked many students in the campus. In fact, the answers varied in different labs. As undergraduate students, we can remain careful when we use the hazardous reagents. But postgraduate students might lower their guard after working in the lab for several years. So we summarized the hazardous reagents most often used in biology labs.
Ethidium bromide (EB) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) to determine DNA and RNA location in agarose gel and SDS-PAGE gel. Due to its ability to intercalate and deform double stranded DNA, EB is considered as a strong mutagen and carcinogen which would impair DNA replication and transcription, as well as to damage mitochondrial DNA during cell proliferation.
Mostly, the amount of EB used in the laboratory (0.25C1 mg/ml) is below the level of toxicity. However, for students under chronic exposure to EB, the potential risk still exists.
EB may evaporate under high temperature (60oC or higher). Thus, EB should not be added into gel when it's not adequately cooled. For protection, nitrile gloves and masks are required.
The disposal of EB remains a controversial subject. Generally, purification treatments are required before the disposal according to different concentrations.
For solvents containing EB over 0.5mM, the solvents should be diluted below 0.5mM and mixed with certain amount of KMnO4 and HCl or NaOH. Low concentrations of EB can be simply mixed with active carbon for at least one hour.
Dimethyl sulfoxide (DMSO) is an important non-proton polar solvent that dissolves both polar and nonpolar compounds. It is widely used in MTT stain and cell preservation.
DMSO is a combustible reagent that must be kept in sealed containers away from heat or flame. Also, it is thought to be toxic for blood vessels, liver and kidney. Also, due to its high affinity with skin, toxins and medicines dissolved in DMSO can be quickly absorbed into human body, which may cause potential side effects, including headaches, burning and itching on contact with the skin.
DMSO can be absorbed mainly through breathing and skin-contacting. For prevention, gloves and eye protectors are required in experiment with DMSO. Common nitrile gloves may provide protection from brief contact but may degrade rapidly in contact with DMSO. Butyl rubber, fluoroelastomer, neoprene, or thick (15 mil) latex gloves are recommended.
Chloroform, or trichloromethane, is a colorless, sweet-smelling, dense liquid that is produced on a large scale as a precursor of polytetrafluoroethylene (PTFE) and refrigerants.
Chloroform may cause irritation to eyes, skin and mucous membrane. Prolonged dermal exposure can result in the development of sores as a result of defatting. It's also a carcinogen and can impair liver and kidney to some extent. Moreover, chloroform dose harm to nervous system that it causes depression of the central nervous system (CNS), ultimately leading to deep coma and respiratory center depression. Serious illnesses would ensue upon ingestion of 7.5 g (0.26 oz) of chloroform. The mean lethal oral dose for an adult is estimated to be about 45 g (1.6 oz). Due to its threat to respiratory and cardiac symptoms, including nausea, vomiting, prostration, jaundice, coma, liver necrosis and degeneration, the anesthetic use of chloroform has been ceased.
Chloroform is not stable and easily decomposed in exposure to light, so it should be kept in brown bottle away from light, heat and acetone (acetone will have acute reactions with chloroform). For protection from chloroform, lab workers should wear proper nitrite gloves and eyes protectors, as well as working in the fume hood. Spent chloroform should be extracted and collected for further disposal.
Tris-(hydroxymethyl)-aminomethane (Tris) is an organic compound extensively used in biochemistry and molecular biology experiments as a component of buffer solutions, such as in Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) buffer, especially for solutions of nucleic acids.
Although Tris is considered as a low-toxic reagent, long-term exposure may still lead to potential threats through skin and breath. Thus, gloves and eye protectors are recommended.
Paraformaldehyde (PFA) is the smallest polyoxymethylene with a typical degree of polymerization of 8C100 units. PFA commonly has a slight odor of formaldehyde due to decomposition. Once paraformaldehyde is depolymerized, the resulting formaldehyde may be used as a fumigant, disinfectant, fungicide, and fixative. The applications of PFA include production of polyoxymethylene plastic (POM or Delrin), root canal treatment, etc. PFA is widely used in organic chemistry labs for various synthetic reactions.
PFA is not a fixative; it must be depolymerized to formaldehyde in solution and kept in sealed containers at a temperature between 2 and 8oC.
It's widely accepted that PFA is a strong carcinogen which is mainly absorbed through skin. It is also irritating to eyes, respiratory system and skin. Thus, lab workers should wear suitable protective clothing, gloves and eye/face protectors and work in fume hoods. In case of contact with eyes, one should rinse immediately with plenty of water and seek medical treatment.
Phenol is widely used in laboratory for its high efficiency in extraction of DNA and RNA. However, this reagent is also highly corrosive to eyes, skin, and respiratory system, leading to burning, allergy reaction or dermatitis. Inhalation or exposure of high concentration of phenol may lead to some diseases in nervous and cardiac system. Besides, long-term exposure to phenol is also toxic to liver or kidney due to both hydrophobicity and the formation of phenoxyl radicals.
According to its toxicity, careful protection is required when operating phenol reagents, such as thick gloves, protection suits and eyes protectors. In case of contact with skin, polyethylene glycol, isopropyl alcohol or plenty of water should be immediately rinsed before seeking for further medical treatment.
Sodium dodecyl sulfate (SDS) is commonly applied in lysing cells during DNA extraction. It is also used in SDS-PAGE to denature protein molecules. Though having little toxicity to the organism, long-term or repeated exposure may still irritate to eyes and skin, or even lead to allergy reaction for certain people. Under this circumstance, protections such as laboratory gloves and eye protectors are still recommended.
Investigating in the lab
In fact we know little about the things actually happening in the labs. So we had to go to many labs and observe the situations for ourselves. We designed a questionnaire and went to observe many biology labs in Tianjin. We talked with the students and got 193 samples for the questionnaire. We did the data analysis and took some pictures in the labs.
All the questions and the results are as follows. To make things clear, pictures taken in biology institutions in Tianjin will also be presented.
Q1. How is the experiment and resting area separated in your lab?
A. There is a separate room for resting purposes
B. Two areas are in the same room on separated benches.
C. Two areas are on the same bench with separated sections.
D. We practically do not separate those two areas.
E. Other situation
There are more and more young scientists returning to China from abroad, which is encouraged by the government. Also, many students choose to seek a higher degree in China and professors are encouraged to supervise more students. Accordingly, many institutes have run out of room for laboratories and the laboratories are sometimes crowded with too many students and equipment. Though many laboratories have set a separate room for resting purposes, many laboratories cannot provide a living room for students. They have to set a special area in the room for living purposes and they agreed not to do experiment near the living area. They will remind each other not doing this. However, some reagents are easy to evaporate and some microorganisms can spread into the air. Things like that would still cause harm to the students. The slight harm can accumulate in the long-term lab work.
Also, there are some labs that don't care about this issue. In some microorganisms labs, the two areas have to share a bench or even do not separate with each other.
Different types of resting area in different labs.
Q2. How frequent do you have meal in lab (excluding the situation if you have the meal in a separated resting room)
A. I had meal in lab almost every day.
B. I often had meal in lab.
C. I only have meal in lab when I have no alternative choices.
D. I have never had and never will have meal in lab.
E. Other situation
Some experiments have special requirements for time management. Sometimes experiments can last for so long that students have little time finishing their lunch. For the labs without a separate room for resting, students might choose to have lunch in the lab. However, most of them know that it's harmful. 38.75% of the students had never had and would never have meal in lab. 30% of the students only do this when they had to.
However, there are still 1/3 of the students unaware of the harmfulness. Usually the leaders of these labs are so strict that they favor students who spend the longest time in the lab. When doing the research, we saw a student having lunch near a mice cage. (The smell is really unpleasant-we wondered how he could do this.) In some cases, students left the snack packages open in the lab while conducting experiments.
We also saw that several labs have a little kitchen in it. In one lab, the "kitchen" is just located beside the experiment bench.
The situation in few labs: the kitchen is really close to the experiment area. The experiment bench is on the left of the shelf while the kitchen is on the right.
Cross analysis: How long have you been working in the lab?
A. Less than 1 year.
B. 1-2 years.
C. 3 years or above.
From this analysis, we could approximately infer that the students would have meal in lab more frequently as they work in the lab for longer time. First, they might become busier and busier. Students are under more and more pressure gradually so they have to spend more time in the lab. Second, they are used to the working environment. As a newcomer, they are vigilant about the dangerous factors. When they are used to the lab, they gradually lower their guard.
Q3. How do you spend weekends and holidays?
A. I spend almost all my time in lab.
B. I often give up my spare time to study in lab.
C. I sometimes give up my spare time to study in lab.
D. I never give up my spare time to study in lab.
E. Other situation please specify
Q4 Approximately how many hours do you spend in lab on weekdays?
Through Q3 and Q4, we could see that students tend to spend most of their time in the lab nearly every day. Many of them work more than 8 hours a day and give up their spare time. In fact, most of them told us that they lack exercise. Though they are encouraged to be hard-working, this habit will show negative influence on their health gradually.
Q5. How do you treat organic waste in your lab?
A. We directly pour them into sewer.
B. We collect them in a special waste liquor bucket.
C. We collect them in a special waste liquor bucket for further treatment.
D. Other situation please specify
Most labs do well when they treat the waste liquor. Most labs have special liquor buckets and use them correctly. Some special labs deal with the waste liquor with the help of specialized companies.
Some labs collect waste liquid in a special bucket for further treatment.
Q6. How do you treat contaminated wastes in your lab?
A. We discard them into a special biological waste bag.
B. We directly discard them after sterilization.
C. We collect them for further treatment without sterilization.
D. We collect them for further treatment after sterilization.
E. Other situation
Q7 How do you distinguish experiment waste and household waste?
A. We do not distinguish them.
B. We strictly distinguish them.
C. Other situation
To protect people outside the lab, experiment waste and household waste should be distinguished strictly. About 15% people didn't distinguish them strictly. We also saw that some people throw the experiment wastes in the bucket containing household wastes. We do really well in our own lab and we think it's a good habit. The two kinds of wastes should be separated in the lab and they will be treated differently outside the lab.
Some labs distinguish experiment and household waste while some labs do not.
Q8. How do you store hazardous and poisonous chemicals in lab?
A. We store them in a special cabinet and cannot use them freely.
B. We store them in a special cabinet yet could use them freely.
C. We store them together with other chemicals.
D. Other situation
Most labs manage the poisonous chemicals strictly. Some labs manage all kinds of chemicals together. In fact, some hazardous chemicals need special management.
Some labs implement strict regulation on hazardous chemicals.
Q9. What kind of personal protective equipment do you usually use?
The things used the most are: lab coats, gloves, and masks. Some labs also use goggles. In fact, there are indeed some students do experiments without any protective equipment. They told us they only wear gloves when using EB. That's a very dangerous habit. This equipment can lower the possibility for people from being hurt by the organisms or the chemicals they deal with.
Some labs require students to use personal protective equipment and put up reminders in the lab.
Initiate a reform in Nankai University
Faculty members are holding a meeting on the topic of hazardous chemical management in response to our advice.
We sent our findings to the president, and he attached great importance to our report. He asked for more details about what we have seen in the laboratories and told us to wait for their response.
Several days later, we received an email from his assistant. She told us that they have held a meeting to rectify and reform dangerous factors in the laboratories. She told us that we helped a lot in this work and the teachers will do their best to strengthen the management of the labs.
Integrating HP into our project
1. The experiment process
Safety has always been an important concern in our laboratory, both in project design and operation during experiment process. This year we used two kinds of Escherichia coli (DH5α in part construction and MG1655 in project). According to the official iGEM guidelines of Risk Groups, E. coli is categorized as a Risk Group 1 organism that "do not cause disease in healthy adult humans" when used properly. Also, the risk group level of E. coli is correspondent with our laboratory safety level (Safety Level 1). To make sure that everything is safe enough, we have seriously managed Individual Safety, Environmental Safety, Biological Safety based on project as well as Safe Shipping.
Individual Safety
1) All members participating in the iGEM project this year have taken safety training courses given by the department experiment center that we acknowledged general safety managements such as fire-fighting strategies, disposition of biochemical materials, proper usages of microwave oven, laminar flow bench and other apparatus.
2) All members have carefully read Safe Project Design, Safe Lab Work, Safe Shipment and other requirements proposed by iGEM Safety Committee. We filled up the Final Safety Form according to the real situation in our laboratory.
3) Nitrile gloves, masks, closed shoes were strictly required in laboratory to prevent potential threats of personal health.
4) Resting area and Experiment area were clearly separated to confine eating and drinking strictly in resting area.
Environmental Safety
1) Biological materials were not allowed to be taken outside the laboratory without sterilization. All of the used materials (bacterial samples, plasmids, reagents, agar, etc.) after experiments were sterilized before leaving the laboratory and collected altogether for further management.
2) Regular cleaning of the laboratory trash was arranged every day to maintain a clean and tidy laboratory environment.
Safe Shipping
We sent our BioBricks through the standard shipping process required by iGEM headquarter.
The design of our project
"Human Practices is the study of how your work affects the world, and how the world affects your work."
—Peter Carr, Director of Judging
iGEM projects involve important questions "beyond the bench" and teams' efforts are often well integrated into the technical aspects of their project, which has a significant influence upon design decisions.
This summer, we had held many activities in education, public engagement, dialogue and biosafety. These human practice activities not only broadened our views on synthetic biology, but also had a significant influence on our project designs.
Check the content below to find out how we have integrated the investigated issues into the design of our project.
1.The topic of our project: Overview
This summer, our team aimed to engineer bacteria for supplement and absorption of autoinducer-2 (AI-2) in the natural environment. We mainly design two cell machines: AI-2 Supplier is the cell machine which can directly supply and enrich the AI-2 molecular level; AI-2 Consumer is another cell machine which can sense, absorb and degrade the AI-2 in the environment. By taking advantage of the special characteristics of AI-2 controllers, we hope to directly control the population behaviors of bacteria in group levels.
What's more, biosafety is further taken into account in our project. By applying gene circuits to control essential gene expression under the assigned biocontainment conditions, we can block essential gene expression to kill the cell upon loss of the biocontainment signal.
2. The interviews in The Ministry Key Laboratory of Molecular Microbiology and Technology: greatly helped us on the execution of our project
In our project, we designed AI-2 Controllers aiming to use these 'controller cells' to modulate the external AI-2 environment. But it's hard for us to apply this project to real world problems. At the same time, we still had some questions on LuxS/AI-2 quorum sensing systems. In order to get professional opinions on our project and to learn more about quorum sensing, we conducted an interview with two specialists, Ms Weixia Gao and Ms Yufen Quan.
Weixia Gao & Yulei Dang
After the introduction of our project idea, we discussed some particularly interesting points. We received advice that helped us to improve our project. This interview was not only helpful and enriching but also led to an agreement to collaborate with the Laboratory of Metabolic Engineering and Environmental Microbiology.
Here are some key points we got from these two specialists:
1. They suggested us to design a response device, which can be an alternative way of testing the AI-2 level in the natural or artificial environment due to the instability of the current measurement method (BB170 test).
2. They suggested us a good way to apply AI-2 Controllers into real world problems: to focus on biofilm. Biofilm formation is a collective behavior in which AI-2 has a significant influence.
After this helpful and enriching interview, we became clearer of our whole project. This interview greatly helped us on the design and actual execution of our project.
3. The dialogue with experts in synthetic biology: greatly helped us on the design of our biosafety system
With the advent of synthetic biology, genetically modified microorganisms are being increasingly used for biomedical, industrial and environmental applications. Deployment of these engineered microbes in large scales and open environments calls for the development of safe and secure means to restrain their proliferation.
Also in the biosafety investigation we took, we found that although most of the labs strictly obey the management rules, there are still some labs with poor management and faint consciousness on biosafety. For example, about 10% of the labs we investigated have no resting area separated from the experiment area. More than 15% of the labs we investigated pay less attention to the treatment of organic waste or materials with engineered bacteria. What's worse, more than 40% of the people investigated would have meals in the lab. These researchers are facing serious safety problems which made us become aware of the importance of biosafety. As the Chinese saying goes, a solid dyke can collapse because of an ant hole in it. Although only a few labs pay poor attention to biosafety, it could still probably cause serious biosafety problems.
After the investigation, we organized many activities to get researcher pay more attention to biosafety. Meanwhile, we were also considering whether our project needed a design to avoid the potential risks of our 'controller cells'.
In order to get professional opinions on our biosafety considerations, we had a dialogue with the experts in this field, Jun Feng. After introducing our investigation and consideration of biosafety, Jun Feng gave us highly appreciation. Also he proposed some suggestions on how to construct a biosafety system.
From the dialogue, we know that traditional metabolic auxotrophy strains are hampered by the potential for inadvertent complementation by cross-feeding or by the presence of the metabolite in heterogenous environments, and synthetic auxotrophy systems rely on extensive genome-wide engineering that may be impractical for many industrial production and biotherapeutic microbes.
An alternative approach to biocontainment is to use gene circuits to maintain essential gene expression or block toxin gene expression under the assigned biocontainment conditions. Upon loss of the biocontainment signal, the circuit blocks essential gene expression or induces toxin gene expression to kill the cell.
After the discussion with Jun Feng, we read plenty of literature about biosafety system design. Finally, we designed a biocontainment system for our AI-2 Controller.
CRISPR/Cas9 method was used to insert mf-Lon tag into 5 essential genes
A schematic of biocontainment system and kill switch.