Safety Form: E.coli and S.cerevisiae

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: Escherichia. 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
     • Source: Buchler Lab and PCR of oligos from IDT
     • Risk Group 1
   • Z4EVpr-iTAL-mCherry
     • Botstein Lab and Addgene
     • Risk Group 2 due to iTAL origin in Xanthomonas
   • Z4EVpr-dCas9
     • Botstein and Gersbach Labs
     • Risk Group 1
   • SNR52pr-gRNA-synthetic binding sequence
     • Gersbach Lab and PCR of oligos from IDT
     • 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 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.
   2. Risks to the safety and health of the general public, if released by 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.
   3. Risks to the environment, if released by design or by accident?
      • No. In the event of exposure of our yeast or E. coli to the environment, there would be no effect.
   4. Risks to security through malicious misuse by individuals, groups, or 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?
   1. Please provide a link to your institution biosafety guidelines.
      • [http://www.safety.duke.edu/BioSafety Duke University Biological Safety Divison]
   2. 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.
      • Yes and our project met their standards for safety
   3. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.
      • [http://www.cdc.gov/biosafety Centers for Disease Control and Prevention]
   4. According to the WHO Biosafety Manuel, what is the BioSafety Level rating of your lab?
      • Our lab has a BioSafety Level rating of 2 due to its ability to handle mammalian cell cultures.
   5. 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.
      • Risk Group 1 and it is below the safety rating of our lab. There is no need for additional safety measures.