Team:Duke/Safety/Ecoli and Scerevisiae

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 (K12) and Saccharomyces Cerevisiae

• DH5 alpha and DBY12397

• Risk Group: 1

2. Highest Risk Group Listed: Risk Group 1

3. List and describe all new or modified coding regions you will be using in your

project.

• TEF1pr-synthetic binding site-YFP

• Z4EVpr-iTAL-mCherry

• Z4EVpr-dCas9

• SNR52pr-gRNA-synthetic binding sequence

o Source: Buchler Lab and PCR of oligos from IDT

o Risk Group 1

o Botstein Lab and Addgene

o Risk Group 2 due to iTAL origin in Xanthomonas

o Botstein and Gersbach Labs

o Risk Group 1

o Gersbach Lab and PCR of oligos from IDT

o Risk Group 1

4. Do the biological materials using in your lab work pose any of the following

risks?

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

2. Risks to the safety and health of the general public, if released by

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

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

the lab?

• No. Our E. coli strain is not virulent and the yeast pose no threat to

humans. The iTALs are derived from Xanthomonas bacteria, but

we did not ever use them as a chassis.

design or by accident?

• No. We are simply aiming to make synthetic gene circuits in yeast

using fluorescent reporters. The already non-virulent E. coli and

yeast are not being programmed to make anything that would

make them virulent or toxic.

• No. In the event of exposure of our yeast or E. coli to the

environment, there would be no effect.

countries?

• No. We are unable to foresee any malicious misuse of our yeast or

E. coli.

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? Also, what

risks might arise if the knowledge you generate or the methods you develop

became widely available?

• Since the expression of the genes of interest is very tunable in

our system, there should be very little problems using one of the

gene circuits in a commercial/industrial manner. If a gene used

in one of these circuits were dangerous if over-expressed. Beta

estradiol could be used to control for this. The entire purpose of

these tunable gene circuits is to give control of the system to the

scientist not the system itself.

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

• As discussed previously, the tunable expression of our promoters

is a design feature of the project that addresses safety risks. All of

the chassis we use are also auxotrophic.

7. What safety training have you received?

• All iGEM team members completed Duke’s General Laboratory

Safety, Chemical Safety, and Fire/Life Safety training before

beginning any lab work

• Information can be found at safety.duke.edu

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

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

b. Does your institution have an Institutional Biosafety Committee, or an

c. Does your country have national biosafety regulations or guidelines?

d. According to the WHO Biosafety Manuel, what is the BioSafety Level

e. What is the Risk Group of your chassis organism(s), as you stated in

• www.safety.duke.edu/BioSafety

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.

• Yes and our project met their standards for safety

If so, please provide a link to these regulations or guidelines if

possible.

• www.cdc.gov/biosafety

rating of your lab?

• Our lab has a BioSafety Level rating of 2 due to its ability to handle

mammalian cell cultures.

question 1? If it does not match the BSL rating of your laboratory,

please explain what additional safety measures you are taking.

• Risk Group 1 and it is below the safety rating of our lab. There is

no need for additional safety measures.