Team:Korea U Seoul/Safety/Safety Questions

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Would any of your project ideas raise safety issues in terms of:

Researcher safety?
Public safety?
Environmental safety?

A. Issues to the researcher safety

Our team uses bacterial genes from Xanthomonas oryzae pv oryzae. Our project covers cloning of RaxR, RaxH, bacteriocin and lysis. We have another project which is constructing a bacterial logic gate. However, bacterial logic gate will not be considered in this part since no experiment was done.

1) Xanthomonas oryzae pv oryzae.

Xanthomonas oryzae pv oryzae is a rod-shaped, round-ended, Gram-negative species. Individual cells vary in length from approximately 0.7 μm to 2.0 μm and in width from 0.4μm to 0.7μm. Cells are motile by means of a single polar flagellum. Colonies on solid media containing glucose are round, convex, mucoid and yellow in colour due to the production of the pigment xanthomonadin, characteristic of the genus. Xanthomonas oryzae pv oryzae is a phytopathogenic bacteria infecting only plants. No research reported that X. oryzae causes human health related risks. Genes (RaxR, H) that we used in this project do not produce toxic chemicals.

2) E. coli (DH5α)

Moreover, E. coli strain we are using is E. coli (DH5α). E. coli (DH5 α) is widely studied and found to be very safe. E. coli (DH5α) cannot survive in the environment; possible expression of growth factor or receptor fragments does not pose an environmental hazard; the vector is non-mobilisable . Thus, E. coli are less likely to harm researcher. Please refer to website provided here to know more about our E. coli strain: http://www.obgyn.cam.ac.uk/cam-only/Safety/GMMO_risk_assessment.pdf

In conclusion, we find our project harmless. Although no significant safety issues were raised during the experiment, it is advised that experimenter should follow safety protocols listed on this website. This includes wearing gloves and goggles as bacteria may cause irritation and skin rash. Please refer to lab safety protocol:
http://kusafe.korea.ac.kr/main.asp (university guideline, Korean)
http://bric.postech.ac.kr/myboard/read.php?Board=safety&id=1481 (national guideline, Korean)

B. Risks to the safety and health of the general public

      There are no significant risks to the safety and health of the general public. First, we do not use pathogenic bacteria. Second, gene products do not induce immune response. Third, our lab follows rules of bio-safety level limiting reagents and material we use. Here are reasons why our project is less likely to cause any health risks.

1) Safety issues regarding chemicals

      Our project, Bacterial Leaf Blight and Bacterial Logic Gate, does not produce any pathogens. In fact, our E. coli (DH5α) strain has no significant hazardous effect. It is widely used among labs, and safety protocols are provided. No known survival mechanism is reported. Furthermore, our gene products, bacteriocin and lysis have no known hazardous effect on human health.

2) Ingestion of the bacteria

      Since we spread the bacteria on rice, it is possible for people to ingest bacteria. However, most of the bacteria will die out before ingestion due to its lifespan or other causes. Most of the time rice is boiled before consumption, so it is not likely to cause serious problem. Nevertheless, complete killing of BLB killing bacteria is recommended. Further research and improvment on our project is required. Please refer to webpage for more information: Project – Improvement – Rice Guardian project

C. Risks to environmental quality

Bacterial Leaf Blight killing bacteria synthesize bacteriocin and lysis as we already discussed. Bacteriocin and lysis genes work only if nearby Xanthomonas oryzae pv oryzae secretes AX21 which is our target protein for detection. However, bacteriocin and lysis are target non-specific. That is to say, once our BLB killing bacteria detect and secrete bacteriocin and lysis, it will attack any bacteria adjacent to Xanthomonas oryzae pv oryzae. This may pose an ecological disruption. Nevertheless, BLB killing bacteria works as ‘suicidal bacteria,’ killing themselves by producing lysis protein eventually. Thus, no further ecological disruption is expected. It is recommended that Xanthomonas oryzae pv oryzae tatget-specific protein be used. Further research is required.
Please refer to our 'further improvement' section for more information: Project – Improvement – Rice Guardian project

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?

Here are BioBricks we used in this project:

In case of "Rice guardian" project, raxR and raxH genes synthesize membrane protein that has no known hazardous effect on human health. Although they are from phytopathogenic bacteria (Xanthomonas oryzae pv oryzae), RaxR and RaxH proteins have nothing to do with phytopathogenecity. They produce protein that detects Ax21. Clearly Ax21 is a pathogenic chemical that infects rice not RaxR and RaxH.
lysis, bacteriocin gene protein has no effect on human physiology. Rather destruction of its own colony is predicted. Maintaining and securing clone product is more important.

3OC6 HSL, 3OC12 HSL, C4 HSL and other molecules used for signaling molecules between gates in binary adder project has no hazardous effect on human. These molecules are used for communication between bacteria and the molecules do not show any hazardous results even to other species of bacteria. The purpose we used genes that code the molecules is that the molecules just fit to conditions required for signaling molecules between gates we designed. The conditions are following : 1. the signals should able to go through membrane quite easily, 2. able to be biosynthesized and 3. existance of promoter activated by presence of signaling molecules.
Also, there are no parts that could directly result critical effects on any organisms in binary adder project.

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?

Regarding local biosafety group

There is no biosafety group, committee, or review board at our institution. The iGEM project has not been reviewed by any local bio-safety committee. However, we follow the bio-safety guidelines for any procedure in the laboratory. Please refer to our safety guideline (includes what biosafety rules we have to consider in our country):
http://kusafe.korea.ac.kr/main.asp (university guideline, Korean)
http://bric.postech.ac.kr/myboard/read.php?Board=safety&id=1481 (national guideline, Korean)

Consideration about biosafety rules or guidelines

      Based on our university guideline and national guideline, we made 3 major biosafety principles and their detailed guideline.

1) Biosafety level of laboratory

      This principle defines boundary of experiment which labs can conducted and limits the type of experiments based on their capability. Each lab should be evaluated for their biosafety capability and assigned any level of biosafety to conduct an experiment. Based on their capability and assigned biosafety level, types of experiments that they can do should be limited. For example, a lab assigned to lowest biosafety level cannot handle pathogenic bacteria.

2) Assessment and evaluation of project and pathogenicity of bacteria

      This principle assess and evaluate biosafety of project and pathogenicity of bacteria. When assessing a project, one should evaluate potential threat for researcher, public, and ecosystem. Like medicinal project undergoing IRB examination before the experiment, biological experiment should go though series of assessment and evaluation. Biosafety committees and scientists should communicate each other and give them thorough feedback.

3) Bio-security

      This principle covers securing biological organism from lost, theft, and misuse. It also deals with regulation after lost, theft, and misuse is happened. To achieve this, bio-security system should be established.

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?

A. Suggestions for safety issue

Safety is the most important part of iGEM competition. Every member of team should know and follow the safety rules. Here are recommendations we give to the future iGEM teams

1) Every member should know about safety rules in the lab. This includes bio-security level, pathogen, gene product, protocol, and etc.

- Each member should know bio-security level of lab. Can lab deal with pathogen? Does the lab have proper equipments for experiment? This kind of consideration is recommended before designing and conducting experiments. Conducting a survey is recommended.
- Each member should be careful if they are dealing with pathogen. How can we prevent disease outbreak? What are safety requirements? Please study about pathogen and related diseases beforehand.
- Each member should consider their gene product. Gene product may be chemically dangerous and may damage ecosystem.
- Each member should be aware of protocols of instruments to avoid any accidents. This is also related to lab results.

2) Communication matters.

Miscommunication leads to accidents and poor results. Results of the experiment should be reviewed and shared by every member of the group. Weekly group discussion is recommended. Each member should know everything about the experiment before one actually does it.

B. How could parts, devices, and systems be made even safer though bio-safety engineering?

-Changing promoter or other regulatory mechanism to shorten the duration of mRNA Changing promoter sequence effectively controls survival time of mRNA and overall production of protein. If mRNA degrades easily, we can control it.
-Adding ‘time bomb’ to kill themselves as time goes. Internal clock bomb will ensure death of our product. So it will be much safer.