The chassis organism we are using for this project.

• E.coli(K 12) DH5α

Highest Risk Group Listed

Risk Group 1

This is a list of our new coding regions in our projects.

Part number	Source of DNA	Species	Risk group	Function
BBa_K2015000	IDT gBlock DNA synthesis	E.coli	1	Self-assembling region (SAR) for the circularization of proteins
BBa_K2015001	IDT gBlock DNA synthesis	E.coli	1	One of self-assembling peptides
BBa_K2015002	IDT gBlock DNA synthesis	E.coli	1	One of self-assembling peptides
BBa_K2015003	IDT gBlock DNA synthesis	E.coli	1	G7 linker contains 7 Glycine
BBa_K2015004	IDT gBlock DNA synthesis	E.coli	1	This part can form disulfide bonds between Sulfur residues for multimerization of protein
BBa_K2015005	IDT gBlock DNA synthesis	E.coli	1	This part can form disulfide bonds between Sulfur residues for circularization of protein
BBa_K2015008	IDT gBlock DNA synthesis	E.coli	1	This part can be formed multimerized protein by using BBa_K2015002
BBa_K2015009	IDT gBlock DNA synthesis	E.coli	1	This part can be formed multimerized protein by using BBa_K2015001
BBa_K2015010	IDT gBlock DNA synthesis	E.coli	1	CDS for circularized protein
BBa_K2015011	IDT gBlock DNA synthesis	E.coli	1	LacI expression unit
BBa_K2015012	IDT gBlock DNA synthesis	E.coli	1	The sequences of downstream can be induced strictly by IPTG
BBa_K2015013	IDT gBlock DNA synthesis	E.coli	1	A part of circularized GFP

Description of the biological materials we are using in the lab.

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

Some materials pose risks to team members. For example, Ethidium Bromide is an intercalating agent so it must be used by with personal safety gear. All lab staff is trained according to safety manual provided by Hokkaido University, to prevent risks. We took on ourselves to compile a shortlist of often used dangerous materials and safety procedures in our project.

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)

• DH5α

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

All genes used in this project come from non-pathogenic bacterial strains of E. coli. Expressed proteins did not show any toxic effect to their host. Our biobricks do not have any foreseeable selective advantage if released to the environment. After consideration we could not find any usage pausing a security concern.

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

Some materials pose risks to the general public. For example, Ethanol is a flammable solution so it must not be used by open fire. Not to release those materials, all lab staff is trained according to safety manual provided by Hokkaido University.

Risks to the environment, if released by design or by accident.

The E.coli strains we use in our lab, are lab sage strains. As a precaution all materials coming in contact are sterilized before and after. Reference Federal Register, (1986) Vol. V1: 88, 6952-16985

Risks to security through malicious misuse by individuals, groups, or countries.

There is no foreseeable risk in misuse of our generated genetic material. Our generated genetic material performs basic functions in biology. However, it is impossible to guard against the incorporation of our parts in malicious settings.

Risks which might arise when our project move from a small-scale lab study to become widely used as a commercial/industrial product.

Our project is about optimizing the expression of genes. Our device does not contain a coding site. Therefore, risk will arise when other users assemble our parts with dangerous coding sites. We have to caution the user when assembling with dangerous coding sites.

Design features to address safety risks.

Our device only contains sequences that regulate the expression of genes. (Promoter, RBS, and terminator) Therefore, our device itself does not contain any safety risks and does not have design feature to address safety risks.

Safety training we received.

We all received a lecture class regarding gene recombination that were held in Hokkaido University. It is based on 'Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms'.

Biosafety provisions

Link to our institution biosafety guidelines.

http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html

Our Institutional Biosafety Committee.

We have a permission to engage in the experiments from the safety officer of genetic recombination of Hokkaido University. All members participating in the experiments are registered with this office. All members are trained according to the safety demands of safety officer of genetic recombination.

Our country's national biosafety regulations and guidelines

Japan is participating in cartagena act.

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
Sone Teruo