1) Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them:

*Species: E. coli (K 12)

*Strain: DH5alpha
*Risk Group: 1
*Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli
*Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

*Species: E. coli (K 12)

*Strain: JM109
*Risk Group: 1
*Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli
*Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

*Species: E. coli (K 12)

*Strain: N99 *Risk Group: 1 *Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli *Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

*Species: E. coli (K 12)

*Strain: N4830 *Risk Group: 1 *Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli *Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

*Species: E. coli (K 12)

*Strain: JM2.300 *Risk Group: 1 *Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli *Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

*Species: E. coli (K 12)

*Strain: MG1655
*Risk Group: 1
*Risk Group Source Link: www.absa.org/riskgroups/bacteriasearch.php?genus=&species=coli
*Disease risk to humans? If so, which disease? : Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

2) Highest Risk Group Listed:

1

3) List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)

* Part name: BBa_K1139100

* Where did you get the physical DNA for this part (which lab, synthesis company, etc): Gene synthesis by IDT.
* What species does this part originally come from?: Arabidopsis thaliana
* What is the Risk Group of the species?: 1
* What is the function of this part, in its parent species?: Produce cytokinin

* Part name: BBa_K1139101

* Where did you get the physical DNA for this part (which lab, synthesis company, etc): Gene synthesis by IDT.
* What species does this part originally come from?: Arabidopsis thaliana
* What is the Risk Group of the species?: 1
* What is the function of this part, in its parent species?: Produce cytokinin

* Part name: BBa_K1139018

* Where did you get the physical DNA for this part (which lab, synthesis company, etc): Kiga lab.
* What species does this part originally come from?: M13 mp18
* What is the Risk Group of the species?: 1
* What is the function of this part, in its parent species?: most of the M13 phage genome

4) Do the biological materials used in your lab work pose any of the following risks? Please describe.

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

>Without drinking large amount of sample, there is no risk. Such taking might cause irritation to skin, eyes, and respiratory tract.

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

>Without drinking large amount of sample, there is no risk. Such taking might cause irritation to skin, eyes, and respiratory tract.

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

>Production of AHL might perturbate communications in wild bacteria.

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

>Use of plant hormone in the opposite direction could disrupt growth of crops. Malicious usage of phage might perturbate intestinal flora.

5) If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available?

>Phage infection in massive scale might increase rate of horizontal gene transfer of antibiotics resistant gene though such transfer also occurs in nature. General use of AHLs will cause crosstalk among cell-cell communication though each project including ours is designed to crosstalk of signals.

6) Does your project include any design features to address safety risks?

>Our chassis, E. coli K12 derivatives except MG1655, are thiamine auxotrophic. A paper, PMID:12949114, also tells growth defects of MG1655, though we will use this strain only in lab.

7) What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.

>Team members have taken classes for bioengineering including recombinant DNA technology.

8) Under what biosafety provisions will/do you work?

a. Please provide a link to your institution biosafety guidelines.

>http://www.rpd.titech.ac.jp/rpdiv/somu/cat71/detail_216.html

b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.

>Our institute has recombinant DNA committee. Our team have applied our experiment plans and received approval from the committee.

c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.

>YES http://www.lifescience.mext.go.jp/bioethics/anzen.html

d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].

>BSL2

e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.

>Group1. It matches the rating of lab.

Faculty Advisor Name:

>Daisuke Kiga