Team:WHU-China/Safety

Brief Answers to the Questions

Welcome to our Safety Page. We will first answer the safety questions asked by iGEM headquarters briefly, and then discuss safety issues associated with our project in detail. In addition, we will provide our ideas and practice on guaranteeing and developing biosafety.

Q1. Would any of your project ideas raise safety issues in terms of: Researcher safety, public safety, or environmental safety?
No. Our design is based on the commonly used E. coli K.12 strain and genes we manipulated are original genes in E. coli. The protein products, at least from current understanding, will cause no harm to researchers, the public and environment. In addition, strict lab practice is executed to further ensure the safety.

Q2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,
Did you document these issues in the Registry?
How did you manage to handle the safety issue?
How could other teams learn from your experience?
Yes, we will discuss this question in latter part of this page (Safety Considerations of Our Biobrick parts and Our Project).

Q3. Is there a local biosafety group, committee, or review board at your institution?
If yes, what does your local biosafety group think about your project?
If no, which specific biosafety rules or guidelines do you have to consider in your country?
Yes. All materials obtained have received the approvals from the department's laboratory management committees. We are also obliged to observe the regulations of Teaching Centre of Experimental Biology and apply for approval for materials before we start our project.

Q4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
Some classified measures should be taken according to the safety of the material. For example, plasmids that may harm the safety, when submitted, should receive more attention and have a stricter package. Some harmful byproducts during experiments should be eliminated properly.
We have done our human practice aiming at understanding attitude of the public towards genetically modified baceria and publicizing biosafety ideas to the public, which we think should be popularized to other teams in future iGEM competitions.

General Safety Issues

In this part, we will illustrate organisms, reagents and equipments we use that may cause safety problems, and introduce our operation standard, management and trains protecting the team members, public and environment.
As above, all the organisms and DNA hosts are not of high individual or community risk. Until now, the only used organism is E. coli K.12 (and some of its varieties, for example, DH5α, a RecA mutated strain). Our current lab of basic-biosafety level 1 is safe enough to manipulate this strain. Only the genome (a generous present from University of Dundee iGEM team), but not the living Salmonella enterica was manipulated in the lab, and the genes from it are homogeneous of E. coli K.12, not associated with pathogenesis. We will not implement our future experimental plan with microorganisms of risk group 2 or vertebrates in our current lab, for too low is the biosafety level and no animal facilities.
To manipulate microorganisms, an ultra clean cabinet is used and strict aseptic technique is followed. All experimenters have been trained on foundation microbiology technique and biosafety. All microorganism contacting vessels are sterilized before and after experiments in appropriate protocols. Also, all microorganism materials will be sterilized before discarded. In this way, we believe that no public or environmental harm will be caused by the experimental organisms. In addition, no one in our team will be hurt by the experimental organisms.
The genetic modifications we make will change metabolism of bacteria. However, there is no evidence both theoretically and experimentally that these modifications will improve the survival capability of the bacteria or cause damage to environment even taking the risk of horizontal gene transfer into consideration. The effects of expressing gene adrA regarding infectivity and pathogenicity of E. coli is not clear now, but it still can be easily controlled in the lab environment without human ingestion. Also, we will insert a death device into the bacteria cell when we construct the whole system (still far from now) to avoid the proliferation out of control. For more information about gene safety, please read the next section and documents of our relevant parts on registry.

We have considered the potential harmful chemicals and equipments, as listed below:

• Basic molecular experiments: NaOH, HCl, SDS, acrylamide, TEMED, ethanol, IPTG, liquid nitrogen, β-mercaptoethanol, xylene cyanol FF.
• Bacteria culture: ampicillin, kanamycin, chloramphenicol.
Chemical analysis and measurements: acetone, cupric acetate, Sudan III, Congo red, Coomassie Brilliant Blue G-250, Coomassie Brilliant Blue R-250.
• Equipments: UV lamp, supercentrifuge, heating equipments (alcohol burner, PCR amplifier, water bath, dry bath), electrophoresis apparatus, -80℃ refrigerator, ultrasonic cell disruptor.

All of these are regular reagents and apparatus in a molecular biology laboratory. The risks of them come from inflammability, explosibility, irritation, corrosivity, toxicity, carcinogenicity and physical injury. But none of them raises special safety issue, with chemical hood, emergency shower, normal personal protection, safety management and safety training.
Only trace amount of antibiotics are used. Inactivated will they be before discared.

Emergency Shower

Safety Considerations of Our Biobrick parts and Our Project

In this section, we are going to answer safety question 2 in detail, taking the potential risk in the future into concern.
The main safety challenge we must face is that as a practical bacteria therapy our direction is, we shall demonstrate that the “E. coslim” will not harm its host when it is developed completely. As few experiments we can do in such limited time, we have done a series of theoretical work to solve problems in this field. Intestine-colonized pathogenic E. coli may cause immune responses, following by diarrhea, inflammation and fever (although the strain we use is considered non-pathogen). Thus we plan to transplant the whole synthetic system to another organism (for example, Bacillus subtilis, has proved safe in human intestine). We know that it is hard as the two organisms are very different on transcriptional mechanisms, but we believe the work of establishing model system (that is what we are doing) in E. coli will make it easier. Also, as the rapid development of synthetic biology and gut microbiology, we hope in the near future, we can modify the genome of E. coli to change it a safe intestine microbe.
Another risk is that when the engineered bacteria get higher efficiency on energy production, they could proliferate out of control. To forestall this situation, we design a “death” device (for more information, click on Death) using D-xylose as inducer. It means if you want to stop weight losing, what you have to do is to eat some D-xylose, and the “E. coslim” will die, shed from the intestinal wall and be poured out. What is more, designed as a two-plasmid system, it prevents all possible horizontal gene transfers.
Further safety issues will be raised and discussed in future experiments, including those operates in intestinal model and animals (For more information, click on Future Perspectives).

Safety Management and Practice

Our project has past a review of an expert committee, of which members are professors or associate professors of microbiology, genetics and bioengineering. Safety issues are considered seriously before they approved our design. We have consulted a few experts for safety questions about both artificial intestinal bacteria and experiments. Their advices help us improve the safety of the whole planning system. For that, we thank Dr. Yulan Wang and Dr. Tiangang Liu so much. Our lab is supervised by Teaching Centre of Experimental Biology, Wuhan University (TCEB, whose leader is our instructor Dr. Zhixiong Xie). An expert group of this centre formulates safety guidelines and guarantees their performance in all teaching labs. All experiments we do past its review. Our safety management system includes the followings: Management of Chemicals, Equipments and Experimenters: Chemicals and equipments are registered in TCEB before they are available for us. Equipments are checked and maintained regular. And an entrance guard system is used to ensure only the admitted could enter the lab.

• Responsibility distribution: All members worked in the lab are divided to three groups. The group leaders arrange schedules of experiments after assessing safety issues in the group (for example, the safety train of the experimental executer). Every member records his/her experiments in detail in the notebook of the group. Each group takes responses for cleaning and checking risks in the lab in turns. The group leader takes responses to the team leader. The team leader reports regular on safety to engineer Miss Long Yan, who is authorized by TCEB and manages the lab.
• Management of Chemicals, Equipments and Experimenters: Chemicals and equipments are registered in TCEB before they are available for us. Equipments are checked and maintained regular. And an entrance guard system is used to ensure only the admitted could enter the lab.
• Training: Our team members have been trained after Guidance of Student Experiments formulated by TCEB.

Guidence of Student Experiments

Biosafety and Publicity

We believe that biosafety is not an issue which should be only considered inside biological lab, but also around people and communities. Spreading knowledge of biosafety to the public will help eliminate misunderstanding and prejudice, from which the science and all the people will benefit. We hope that sharing this idea with all iGEM teams can be useful to take advantage on biosafety.
To investigate public attitude towards our project and to publicize our biosafety ideas are what we do for our human practice. As “eating bacteria” is somehow an unacceptable concept now, we would like to find whether people know the truth about biosafety issues raised by bioengineered bacteria, or they are just panicked by their imaginary “bacteria”. And then, after initiating basic knowledge of microbiology, gene engineering and synthetic biology, we shall take a look on if people’s opinion to biosafety issues will change. In this way, we will estimate the value of our scientific publicity.
The rational discussions of biosafety we take with the public do not limited on our project, but also hotspot issues such as transgenics and stem cell therapies. For more information, please click on Human Practice.

Introducing a Simplified Intestinal Model