Safety form 1

a. 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:

A: We use e.coli(DH5α), which is in Risk Group 1; the link is ; Disease risk is that it may cause diarrhea, if used carelessly, just as the wild type of E.coli.

b. 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.)

A: We use dCas9 and sgRNA as our new parts. We bought the plasmids carrying these elements from Addgene and got them from one of our advisors. They are from Streptococcus pyogenes originally, which belongs to Risk Group 2. They all function as the major parts of CRISPR system.

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

A: It may cause diarrhea, if used carelessly, just as the wild type of E.coli; and it may introduce antibiotics resistance into environment.

d. 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? (Note: This is meant to be a somewhat open-ended discussion question.)

A: The E.coli may release to other labs, thus competing with the strains of E.coli which are used by those labs. It might affect the producing efficiency in fermentation engineering process if being used widely.

e. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.

A: No, because we believe the new BioBrick parts we made this year do not raise any safety issues.

f. 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.

A: We use agents and culture that are most generally applied and exclude any infectious host organisms. researchers are all required to wear protective gears such as glasses, masks, lab coats and double-layered gloves.

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


h. 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.

A: No, we do not have an Institutional Biosafety Committee.

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


j. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab?

A: 3

Safety Form 2

a. Organism name and strain name or number.

A: Streptococcus pyogenes, it is in RG2

b. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.

A: We use Cas9 and sgRNA parts originally from this organism. These 2 parts compose the CRISPR system, which helps it to cut the exogeneous DNA and functions like an immune system or RNAi. This year Cell publishes a paper and the researchers change Cas9 protein into dCas9, which can specificly knock down gene expression level together with sgRNA. So we consider this new genetic tool as a perfect candidate for us to use in order to change the reporter gene expression level to certain ammounts via an easy and convenient way.

c. How did you physically acquire the organism or part?

A:The dCas9 protein is bought from Addgene company and the sgRNA is from the lab of Dali Li, one of our advisers, in East China Normal University.

d. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?

A: There are no potential safety or health risks to all the people.

e. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?

A: Cells after transformation will be carefully dealt with and all of processes will follow the rules and regulations of the lab.

f. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.

A: Both dCas9 and sgRNA are parts of a novel genetic tool originated from CRISPR system, which draws more and more attetion from a lot of researchers nowadays. Their only function is to knock down specific genes, in our project the reporter genes, to a certain level. The mechanism is that dCas9 will bind to specific DNA sequence with guidance of the specific sgRNA and block RNA Polymerase to prevent the transcription process. All of these details have been studied by scientists and during the whole process there will be no risks aroused to human beings and the environment.

g.Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?

A:This system offers us a very easy and convenient way to regulate different genes simultaneously and independently. As far as we know, only this system can match our goal due to its novelty and simplicity.

h.Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?

A:Our parts are not restricted for transport and they meet all of the requirements.

i. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.

A: The safety level of our lab is 3 and our experiment is performed in a safe way.