Nagahama

Safety Form

Your Training

a)Have your team members received any safety training yet?

Yes, we have already received safety training.

b) Please briefly describe the topics that you learned about (or will learn about) in your safety training.

Our country ratify the law concerning the conservation and sustainable use of biological diversity through regulations on the use of living modified organisms. So we have learned the law of recombinant DNA experiment in Japan, the difference of experimental conditions required for various biological species, the method for sterilization of living modified organisms, and the rule for starting a new recombinant DNA experiment in our institute.

c) Please give a link to the laboratory safety training requirements of your institution (college, university, community lab, etc). Or, if you cannot give a link, briefly describe the requirements.

Sorry, our institute doesn't open the laboratory safety training requirements to the public. In our institute, reseachers and students who are planning to start a new recombinant DNA experiment, must receive a lecture about recombinant DNA experiment that are held by our genetic modification safety committee before starting the new recombinant DNA experiment.

Your Local Rules and Regulations

a) Who is responsible for biological safety at your institution? (You might have an Institutional Biosafety Committee, an Office of Environmental Health and Safety, a single Biosafety Officer, or some other arrangement.) Have you discussed your project with them? Describe any concerns they raised, and any changes you made in your project based on your discussion.

We have a genetic modification safety committee in our intitute, and our team primary instructor is a member of the committee. Our project have been discussed and permitted by the genetic modification safety committee. There was no concern and change during the discussion.

b) What are the biosafety guidelines of your institution? Please give a link to these guidelines, or briefly describe them if you cannot give a link.

Sorry, our institute doesn't open the biosafety guidelines to the public. Brieafly, reseachers and students who are planning to start a new recombinant DNA experiment, must receive a lecture about recombinant DNA experiment that are held by our genetic modification safety committee, and permission of new experiment before starting the new recombinant DNA experiment. In our institute, only P1, P1A, P1P and P2 experiments can be permitted.

c) In your country, what are the regulations that govern biosafety in research laboratories? Please give a link to these regulations, or briefly describe them if you cannot give a link.
Biosafety in Japan

http://law.e-gov.go.jp/htmldata/H15/H15HO097.html (Japanese version)

http://eiyaku.hounavi.jp/eigo/h15a09701.php (unofficial English version)

MEXT LifeScience Portalsite

http://www.lifescience.mext.go.jp/bioethics/index.html (Japanese version)

http://www.lifescience.mext.go.jp/english/index.html (English version)

The Organisms and Parts that You Use

File:Nagahama Safety2017 Spreadsheet.xls

Risks of Your Project Now

Please describe risks of working with the biological materials (cells, organisms, DNA, etc.) that you are using in your project. If you are taking any safety precautions (even basic ones, like rubber gloves), that is because your work has some risks, however small. Therefore, please discuss possible risks and what you have done (or might do) to minimize them, instead of simply saying that there are no risks at all.

a)What is your chassis organism?
E. coli K-12 JM109
Saccharomyces cerevisiae BY4742, CEN.PK 113-6B

b)Do you plan to experiment with any other organisms, besides your chassis?

In order to make production of beta-carotene by S. cerevisiae, we will insert carotenoid biosynthesis genes crtE, crtYB and crtI derived from X. dendrorhous on the genome of S. cerevisiae by homologous recombination. We use sod2 from S.pombe to give Saccharomyces cerevisiae salt tolerance.

c) Risks to the safety and health of team members, or other people working in the lab:

We used nucleic acid which TEF2 gene promoter region and HMG1 from S. cerevisiae, sod2 gene from S. pombe and crtYB/I/E from Xanthophyllomyces dendrorhouse.

The DNA sequence of each gene has already been determined, and its gene product does not show properties such as pathogenicity, toxicity, carcinogenicity, drug resistance and the like.

HAA1 overexpressed by the TEF2 gene promoter is a transcriptional activator involved in adaptation to weak acid stress and activates transcription of genes encoding TPO2, YRO2, and membrane stress proteins.

sod2 encodes the Na + / H + antiporter gene, giving the host Na + efflux ability and salt tolerance. HMG1 encodes HMG-CoA reductase and catalyzes conversion of HMG-CoA to mevalonate, which is a rate-limiting step in sterol biosynthesis.

Bifunctional enzyme ,crtYB, catalyzes the reactions from GGPP to phytoene (phytoene synthase) and lycopene to beta-carotene via the intermediate gamma-carotene (lycopene cyclase).

crtI is enzyme involved in Carotenoid biosynthesis.crtI catalyzes conversion from phytoene to lycopene via the intermediate of phytofluene, ζ-carotene and neurosporene by introduction of four double bonds.

crtE catalyzes the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) to produce geranylgeranyl diphosphate (GGPP) which needs for carotenoid and diterpene biosynthesis.

Because the hosts ,E. coli and S. cerevisiae, has no toxic to human, there may be no risk to human safety. All engineered E. coli and S. cerevisiae have been sterilized by autoclave sterilization. We use hexane to collect β-caroten from S. cerevisiae culture. Hexane is flammable. These solvents are not used beside the fire. We have worn rubber gloves and have sterilized all wastes to reduce the risks, including safety level 1 procedures.

d) Risks to the safety and health of the general public (if any biological materials escaped from your lab):
We only use E. coli JM109 and S. cerevisiae BY4742. These organisms belong to Risk group 1. So we think that these organisms have no risks to the safety and health of the general public.

e) Risks to the environment (from waste disposal, or from materials escaping from your lab):
We are using only E. coli JM109 and S. cerevisiae BY4742. They belong to B1 level in biological safety in Japan, and can not live in nature. So if they escape from our lab, there is no risk to the environment.

f) Risks to security through malicious mis-use by individuals, groups, or countries:
All E. coli, S. cerevisiae and DNA parts are stored in private refrigerators for our project, and the refrigerators have been locked when we do not use them.

g) What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.)
As described above, we only use B1 level E. coli and S. cerevisiae in our project and all biological materials are autoclaved before waste according to our institute rule.

Risks of Your Project in the Future

What would happen if all your dreams came true, and your project grew from a small lab study into a commercial/industrial/medical product that was used by many people? We invite you to speculate broadly and discuss possibilities, rather than providing definite answers. Even if the product is "safe", please discuss possible risks and how they could be addressed, rather than simply saying that there are no risks at all.

Q) What new risks might arise from your project's growth? (Consider the categories of risk listed in parts a-d of the previous question: lab workers, the general public, the environment, and malicious mis-uses.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? Does your project currently include any design features to reduce risks? Or, if you did all the future work to make your project grow into a popular product, would you plan to design any new features to minimize risks? (For example: auxotrophic chassis, physical containment, etc.) Such features are not required for an iGEM project, but many teams choose to explore them.
In our project, we have used S. cerevisiae BY4742 strain encoding auxotrophy, which cannot grow in natural field. In future use, we will try to create yeast which make necessary nutrients for nation which is troubled by starvation while have resistance to high salt concentration and low pH.The engineered organisms will be used under supplication of required nutrition, so they cannot grow outside.

This project have the risk that we eating recombinants. We must need to sterilize recombinants before eating.We thought killing recombinants to heat.