Would the materials used in your project and/or your final products pose:

a. Risks to the safety and health of team members or others in the lab?

The chassis being modified is Escherichia coli (E. coli) K12 strains BL21, a level 1 biosafety containment agent. E. coli is one of the most researched gram-negative bacteria of all time. Not only does it have no known survival mechanisms outside the laboratory, the organism is considered to be safe and practical for research. Therefore, there could only be minimal harm inflicted to most entities. One exception to this rule is horizontal gene transfer. By additional transformation, transduction, bacterial conjugation, or gene transfer agents, the bacteria can become more dangerous. Even though this can be said for most parts, the products from our GEO are not dangerous and we have also designed a safety module to reduce horizontal gene transfer.

Other than the common substances used in the process of genetically engineering E. coli, we also experimented with paraformaldehyde, tetracyclin, and sulfonamide. From the list, only paraformaldehyde is of upmost concern. This substance is a carcinogen and has a toxic level of 7. However, the chances of danger is slim as all work concerned with paraformaldehyde is generally completed in fume hoods and proper safety precautions are followed at all times. Paraformaldehyde is also substituted later on with safer substances.

b. Risks to the safety and health of the general public if released by design or accident?

Our GEOs are modified to express GBP and SCFV on the outer membrane of E. coli K12 strains, which should not have the potential to harm the public if released by design or by accident. This is mainly because we can only use the limited resources allowed by biosafety level 1. In addition, E. coli K12, tetracycline, ampicillin, and sulfonamide can be acquired by the public. Unless there is a wide spread of chloramphenicol resistance to harmful bacteria, there is barely any risk to the general public. This is due to chloramphenicol being an antibiotic used in the selection process. In addition, all experiments involving live E.coli are generally carried out in laminar hoods to prevent any unnecessary GMO (Genetically Modified Organisms) from being released. Furthermore, the laboratory has restricted access to non-personnel and proper waste disposal is followed as well as personal hygiene. For example hand washing with disinfectant prior to leaving the lab to prevent spreading of potentially hazardous substances to the public or environment, especially to immunoincompetent and immunocompromised beings.

c. Risks to environmental quality if released by design or accident?

E. coli K12 has no survival mechanism in the environment and cannot spread spores or reproduce in the gut of an organism, thereby proving to be a low environmental threat. Furthermore, a biostatic safety module is created to reduce horizontal gene transfer and paraformaldehyde is replaced later on. Therefore, there are no unreasonable effects to the environment as we strictly comply with the standard waste disposal system. For example, all contaminated waste is considered as “Bio-hazardous Waste” and is disposed in a sealed waste bin after treated. In addition, we are studying the effects when users do not follow the proper waste disposal guide to improve our project.

We believe extensively in the three 'R's - Reduce, Reuse and Recyle. By considering these constraints, we have learnt to plan our experiments better.

Even though it is it is generally assumed that E. coli would be out-competed by natural strains once it is outside of a lab, we assume the fact that in case of accidental release of any GEB, it would raise safety issues because we don't know the potential effects. So any Genetically Engineered Bacteria (GEB) can be potentially dangerous if released in the environment, either on purpose or by accident. The concern about Horizontal Gene Transfer or spread of GEB lead us to develop this project to protect the environment from synthetic devices. During the work on our Biosafety system, we protected the environment from contamination by waste products: all hazardous waste was placed in the correct container (e.g. biohazard containers for biological waste such as E. coli colonies), autoclaved and disposed of responsibly by the university. Team members were taught proper molecular biology skills and aseptic techniques. Team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.

d. Risks to security through malicious misuse by individuals, groups or states?

No. The genes we are inserting into the E. coli are not in themselves dangerous. However, if merged with other parts and components, it would be possible to construct a hazardous organism. Nevertheless, this can be said to most synthetic biology parts. For prevention of HGT, we are studying the strength of bacteriostatic agents. Since we want the bacteria to be able to perform but not able to reproduce. This would reduce the chance of others using our GEO for malicious practice. Furthermore, misuse of bacteriostatic agents is the same as drug abuse. Therefore, there is no addition danger due to the invention of our product.

Specifically, are any parts or devices in your project associated with (or known to cause) pathogenicity, infectivity, or toxicity, threats to environmental quality, or security concerns?

There used to be a carcinogen that was part of project safety consideration. However, due to its level of toxicity we replaced it with safer and more accessible bacteriostatic agents. Unless our GEO is modified to be pathogenic there is barely any chance of infectivity. The reasons being: (1) they are normal occurrence in the human gut; (2) can support their hosts by generating Vitamin K2; (3) can inhibit the formation of additional pathogenic bacteria within the intestine; (4) the E.coli strains in the lab are non-pathogenic.

If your response to any of the questions above is yes:
a. Explain how you addressed these issues in project design and while conducting laboratory work.

To address the issue of additional horizontal gene transfer (HGT), we did numerous experiments with bacteriostatic agents in order to discover the best combination with K12. We tried to determine the optimum time, optimum concentration, and best compatibility between the chloramphenicol resistant E. coli and the bacteriostatic agents. In addition, there are no real environmental threats due to the lab being BSL 1 (limited supplies) and the public having full access to the same materials.

b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

Did you document these issues in the Registry?

As previously mentioned, biosafety is an important aspect of our project as we try to be as people and environmentally friendly as possible. The risk of dissemination of antibiotic resistance is a concern as well as the nonspecific documental of parts. Therefore, we will put in our best efforts to document each and every issue in the Registry.

How did you manage to handle the safety issue?

There were a couple of proposals by the team: (1) sealed containment to avoid the release of GMOs; (2) create a kill-switch and degrade the genome; (3) reduce the rate of reproduction.

How could other teams learn from your experience?

We believe that by discovering the best bacteriostatic agent compatible with chloramphenicol resistant E.coli, we can create better, innovative projects. No longer are we dependent on kill switches or microfluidics, we can have “zombie” bacteria. They are not considered “alive” because they cannot reproduce yet still maintain their function. Nevertheless, teams should know that if there is no innovative spark on creating a new mechanism, there are always the existing mechanisms.

Under what biosafety provisions will / do you operate?

Our laboratory at National Chiao Tung University operates under the restrictions and rules of Biosafety Level 1. On the other hand, majority of teams have a higher BSL. This permits them to possess more dangerous substances, and thus have additional safety and security regulations to follow.

a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.

National Chiao Tung University has its own Environmental Protection and Safety Center. The center was created to have trained personnel oversea the entire university students’ safety when working with hazardous substances. Since the Environmental Protection and Safety Center is one of the subsidiaries in Taiwan, the biosafety rules that govern NCTU is not considered institutional based as most universities are over seen by the same center.

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? Describe any concerns or changes that were made based on this review.

There is a biosafety committee at NCTU and we have conferred each and every aspect of our sensor with them in addition to our instructors, advisors, and biosafety officer. They found paraformaldehyde a bit dangerous so we reiterated that we are no longer using this substance in the finished product. In addition, they find the concept of using bacteriostatic interesting and look forward to seeing our completed project. Before the start of the iGEM project, we received approval from the Principal, our department head, and numerous other sectors of our school.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

Before starting any experiment, all members participated in and passed a series of laboratory safety courses held by Environmental Protection and Safety Center http://esc.nctu.edu.tw/ . The safety module include general lab and occupation safety, biosafety in the laboratory, and general biohazard courses. These courses educated and tested participants about general biosafety principles, knowledge of proper conduct, dressage, emergency response and procedure, waste disposal, etcetera.

After finishing the biosafety training module, we started our lab training with advisors and instructors, Professor Chen and Professor Lee. They taught us from the basics of synthetic biology and then demonstrated specific methods and processes we need to use to isolate plasmid, cut restriction sites, ligate genes, and transform the DNA used to create our bacterium twice before letting us operate.

d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

4. OPTIONAL QUESTION: Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Our team wants to use both existing and novel mechanisms to help create the ultimate solution. However, due to the time constraint, we were only able to experiment with bacteriostatic agents. The experiment that we were hoping to accomplish, but never started is the use of containment or kill switch (destroy non-engineered plasmid that fills the E.coli) versus the use of proteorhodopsin construct, pSB1Pc (BBa_K572100) created by a past iGEM team (aid cell growth and provide a selective pressure in a limited nutrient media). We hope that fusing the best existing mechanism with our recommended bacteriostatic agent can help generate a starting point for the ultimate solution.