Basic Safety Questions for iGEM 2013

The organisms and parts that we use

E.coli JM109 1 BBa_K1524100　Synthesised, Sigma Alderich E. coli 1 stem-loop BBa_K1524101 restribution kit E. coli 1 Reporter gene BBa_K1524102 restribution kit E. coli 1 Reporter gene BBa_K1524104 Synthesised, Sigma-Genosys E. coli 1 sense fragment BBa_K1524105 Synthesised, Sigma-Genosys E. coli 1 sense fragment BBa_K1524106 Synthesised, Sigma-Genosys E. coli 1 sense fragment BBa_K1524107 Synthesised, Sigma-Genosys E. coli 1 sense fragment BBa_K1524108 Synthesised, Sigma-Genosys E. coli 1 sense fragment >Description of the biological meterials we are using in the lab. Dangerous chemicals Chloroform - corrosive and toxic: must be used in fume hood Ethidium Bromide - intercalating agent: must be used with personal safety gear Ethanol - flammable: must not be used near open flame or in large quantities Liquid Nitrogen - cryogenic container and cryogenic gloves must be used Procedures and equipment Agarose gel production - heating in sealed container (rupture risk), scalding hot and vicious during preparation (burn injury risk) - remove container lid before heating in microwave, use safety gear, wait for a few moments before removing from microwave Benson burner - fire risk: DO NOT use flammable materials especially ethanol near open fire Centrifuge - high velocity: balance appropriately, observe the machine till it reaches top velocity Autoclave - high pressure: check the water level, DO NOT open when pressurized UV radiation - damage to eyes and skin: use glove and UV box or UV shield Non-pathogenic bacteria (policy requires treating as pathogenic, as precaution) JM109 DH5alpha Both of these are lab safe strains. As a precaution all materials coming in contact are sterilized before and after. Reference Federal Register, (1986) Vol. V1: 88, 6952–16985 Safety equipment Gloves Coats Goggles UV Box UV shield Waste disposal and sterilization All equipment and waste coming in contact with bacterial is sterilized by autoclave or bleach. All chemicals compounds were disposed according to requirements for their disposal. All table surface used for work were sterilized with 70% ethanol before and after a procedure Chemical Usage All chemical compounds were used according to their manuals and respective material safety data sheet > Genetic material Risks to the safety and health of team members, or other people working in the lab: All lab staff is trained according to safety manual provided by Hokkaido University. We took on ourselves to compile a shortlist of often used dangerous materials and safety procedures in our project. Risks to the safety and health of the general public (if any biological materials escaped from your lab): Device which we make, will not code odd protein. There is no risk by themselves. Risks to the environment (from waste disposal, or from materials escaping from your lab): Biodevice which we make, will not code odd protein. There is no risk to environment. Risks to security through malicious mis-use by individuals, groups, or countries: Our project is about improving antisense RNA system to be useful. They don't code odd proteins. What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.) Our projects are only related to antisense RNA system. They are not toxic substances nor substances that influence nature. Therefore that designs are not need. >Risks of Your Project in the Future Our project aims to make silencing system more efficient by using antisense RNA. There are no risks because the system leads to only control expression of proteins. Safety training we received. We all received a lecture class regarding gene recombination that was held in Hokkaido University we belong to. It is based on 'Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms' , 'Regulations related to the Enforcement of the Law concerning the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms' and 'The Ministerial Ordinance Providing Containment Measures to Be Taken in Type 2 Use of Living Modified Organisms for Research and Development' , Japanese laws. >Biosafety provisions Link to the laboratory safety training requirements of our institution. http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html#e000000477 (Biosafety guidelines of Hokkaido university, section 5-23) http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html (Biosafety guidelines of Hokkaido University) Our country’s national biosafety regulations and guidelines http://www.bch.biodic.go.jp/houreiList06.html http://www.bch.biodic.go.jp/houreiList04.html http://www.bch.biodic.go.jp/houreiList01.html (Reference Ministry of the Environment) >Biosafety Level rating of our lab Our labs Bio safety level is 2. >The Risk Group of our chassis organisms The Risk Group of our chassis organisms is 1. >Faculty Advisor Yamazaki Ken-ichi

Part number	Source of DNA	Risk group	Function
E.coli	DH5α	1	Plasmid