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From 2013.igem.org

*Safety forms were approved on 9/18/13 by David Lloyd and Julie McNamara.

Safety Considerations

According to the American Biological Safety Association, the host bacteria that we used over the summer (Top10 Invitrogen E. coli cells), is at risk level 1, the lowest level possible. Therefore we know that if the bacteria somehow were to make its way outside the lab, it would pose very little harm to the wider public. We also were dealing and handling with pH-inducible promoters, a constitutive promoter, and GFP this summer. None of these parts are dangerous or toxic to the environment.

When we worked with dangerous chemicals during the summer, such as Ethidium Bromide, we disposed of said toxins in appropriate waste bags that would be disposed of in the correct manner.

Although we were working with E. Coli cells this summer, our final vision involved having our construct be functional within bacteria that are natural to the oral biome of humans. That is why we believe that the final product that will result from continued experimentation will be safe for human use.

Safety Questions

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)
• Strain: Invitrogen Top10
• Risk group: 1
• Risk group source link: www.absa.org/riskgroups/bacteriasearch.php?genus=Escheria&species=coli
• Disease Risk to Human: Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

2. What is the highest risk group listed

It is 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.)

• Asr-RBS Promoter // Source: PCR from Invitrogen E. Coli. Genomic DNA // E.Coli // Risk Group: 1 // Fn: pH Responsive Promoter
• GadA-RBS Promoter // Source: PCR from Invitrogen E. Coli. Genomic DNA // E.Coli // Risk Group: 1 // Fn: pH Responsive Promoter
• Lpp-RBS Promoter // Source: Purchased from IDT // E.Coli // Risk Group: 1 // Fn: Constitutive Promoter
• BBaE0040 // Source: PCR from Kit Plate 5 Well 14K // Aequeora Victoria // Risk Group: 1 // Fn: Fluoresce green light after absorbing blue light

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?

No. The strain of E. Coli we used to extract the promoters for Asr, GadA, and Lpp is non-virulent. We also obtained GFP from E. Coli and not from Aequeora Victoria itself, so we did not have any contact with the jellyfish.

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

No. Our project does not raise safety issues in terms of public safety. While the goal of the project is to design a system to counteract cavity formation in the mouth, the actual risks of exposing bacteria to the human digestive system are extremely low. In addition, people consume bacteria on a daily basis.

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

No. In the event of a spill, our engineering organism doesn't produce any toxic substance and should exhibit a low environmental safety risk.

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

No. We cannot foresee any possible misuse of our engineered organism by anyone. The risk of misuse is minimal since the bacteria, in its current form, only produce green fluorescence protein.

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?

One might replace the GFP with toxic substances, but this risk is no greater than regular, non-engineered bacteria. The dual state promoter, in itself also pose no greater threat than a single state promoter since it only helps up-regulate the expression of certain genes.

6. Does your project include any design features to address safety risks? Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.

Our product does not actually switch on above a basal level until the pH is low since both ASR-RBS and GadA-RBS promoters are induced by acidic conditions.

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.

Our instructors demonstrated proper lab safety procedures at the inception of our project. This includes proper disposal methods of biohazardous waste, proper use of ehidium bromide, emergency eye-wash and body-wash techniques, and more.

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

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

http://www.research.northwestern.edu/ors/safety/biological/biosafety-manual/biosafety-levels.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.

The Northwestern University Institutional Biosafety Committee (IBC) work together to ensure safe and ethical research practices in the Northwestern community. We did not discuss our project with the IBC, but we worked under the supervision of advisors whose labs have received IBC approval

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

Yes. The following is from the Centers for Disease Control (CDC): http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features.

Our lab is Biosafety Level One.

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.

The organisms match the safety level of the lab.

Faculty Advisor Name

Dr. Joshua Leonard & Dr. John Mordacq