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1. Would any of your project ideas raise safety issues in terms of:
   • researcher safety,
   • public safety, or
   • environmental safety?

We are transforming genes into non-pathogenic Escherichia coli and Zymomonas mobilis, which are biological safety level 1 organisms, using plasmids with various antibiotic resistances. Furthermore, we are attempting to isolate bacteria from the environment capable of degrading lignin, alginate, and polystyrene.

Researcher Safety

While most of our project is in lab strains of E. coli (NIH Risk Group 1), we are culturing organisms from environmental samples that are able to survive in minimal media containing one of compounds lignin, alginate, or polystyrene we are hoping to degrade. Due to immediate culturing in minimal media, it is unlikely that we will culture any significantly pathogenic organisms from our turtle pond on campus, according to the Caltech Safety Office.

We are then transforming the genes capable of degrading those compounds into E. coli, using standard laboratory procedures. The strains are also non-pathogenic.

Once the bacteria degrades the compound, we then use bacteria that can eat the degraded polymers. These E. coli ferment and create ethanol, a form of bio-fuel. The strains we are using are not dangerous, but ethanol in high concentrations can pose a potential health hazard. Nevertheless, we are not producing ethanol in high enough concentration for major concerns.

Public and Environmental Safety

Because we isolated our organisms from a healthy environment, there is not much to worry about in terms of public safety. Our strains of E. coli are also typical laboratory strains, the bacteria is unlikely to survive in the environment. Nevertheless, we make sure to follow the bio-safety regulations for the engineered microbes, such as bleaching the cultures before disposing them. Our strains are not meant to be released into the wild.

In the case that some cells do escape into the environment, a method of containing genetically modified bacteria is to prevent the recombinant plasmids from being taken up by native species (horizontal gene transfer) and to prevent the engineered strain from outcompeting the local community. A common method is to use the ccdB gene ([http://partsregistry.org/Part:BBa_P1016 BBa_P1016] and [http://partsregistry.org/Part:BBa_P1010 BBa_P1010])on a plasmid in conjunction with an E. coli strain such as DB3.1 ([http://partsregistry.org/Part:BBa_V1005 BBa_V1005]). The plasmid will code for the “death gene” which will kill any cell that does not code for immunity in its genome. If a native microorganism would uptake this man-made plasmid, it would die, preventing the propagation of the recombinant DNA in the environment (Featured Parts: Cell Death).

Another similar “suicide” containment system uses streptavidin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J36841 BBa_J36841]) (Kaplan, Mello et al. 1999; Urgun-Demirtas, Stark et al. 2006). This protein binds very tightly to biotin, a required co-enzyme for many metabolic pathways. This makes biotin unavailable and causes cell death. Kaplan et al. reported cell counts were reduced 99.9% in eight hours after their system was activated by absence of pollutant to degrade.

None of the above is necessary because normal lab strains are engineered so that they will be unable to survive in the field.

 

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,
   • did you document these issues in the Registry?
   • how did you manage to handle the safety issue?
   • How could other teams learn from your experience?

Our new BioBricks parts consist of genes

 

3. Is there a local biosafety group, committee, or review board at your institution?
   • If yes, what does your local biosafety group think about your project?
   • If no, which specific biosafety rules or guidelines do you have to consider in your country?

Yes, Caltech has an Environment, Health, and Safety (EHS) Office. Our team completed safety training, including a walkthrough of the lab with the lab safety officer before beginning any lab work. We also spent about 3 days with our graduate student mentors learning molecular biology techniques and the safety measures associated with them. We talked to the Caltech safety office about our project. They saw no problems with our handling of biological safety since we are working with level 1 organisms. They noted that the possibility of growing pathological bacteria from our enrichment cultures is low due to immediately diluting in minimal media rather than growing environmental samples up on LB. If we suspect any of the cultured organisms are harmful, we will work with the safety office to implement proper safety procedures. Furthermore, we talked to the safety office about possible chemical hazards in our lab. Any solid waste (tubes, etc) should be put in a container or bag and also labeled as hazardous waste.

Safety Regulations

International:

• [http://www.who.int/csr/delibepidemics/WHO_CDS_CSR_LYO_2004_11/en/ WHO]
• [http://bch.cbd.int/protocol/ Convention on Biological Diversity]

National:

• [http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm NIH]
• [http://www.cdc.gov/biosafety/ Center for Disease Control (CDC)]
• [http://www.absa.org/ American Biological Safety Association]
• [http://www.epa.gov/opptintr/biotech/index.htm US Environmental Protection Agency (EPA)]

Local:

• [http://safety.caltech.edu/ Caltech Safety Office]
• [http://safety.caltech.edu/committees/committees_biosafety Caltech Institute Biosafety Committee]
  • [http://safety.caltech.edu/documents/24-biosafety_manual.pdf Caltech Biosafety Manual (pdf)]
  • [http://safety.caltech.edu/documents/22-biohazardous_agent_classification.pdf Caltech Biohazardous Agent Classification (pdf)]
    
    

How we comply with Institute Guidelines

Training

Before working in the lab, we were required to attend the SURF Safety Presentation, a talk given by the Caltech Safety Office that goes over general guidelines for working in any sort of lab.

• How to assemble if there is a fire
  • For Braun, outside by Wilson Ave, letting the floor warden know if everyone from your lab evacuated
• Shelter in place for earthquakes and other hazards
• Emails, phone calls and texts through the automated message system will alert everyone to threats on campus
• Wear appropriate attire to work
  • Closed toed shoes
  • Long pants if working with large amounts of dangerous chemicals
  • Avoid working alone and let someone know that you're in lab if you must work alone
• For emergencies, call security at ext 5000.

The safety officers for Braun 16 are Dr. Michael Vicic, Linda Song and Grayson Chadwick. They gave us a tour of the lab, showing us were safety equipment was before getting card access to the building and lab.

• Shower and eyewash located near the main door
• Fire extinguisher near main exit
• First aid kit at front of room
• Telephone near main exit

Our graduate student mentors, Emzo de los Santos, Nate Glasser and Gita Abadi gave us a 2-day bootcamp before we could begin work on the project. This included how to perform sterile techniques, operation of the equipment we would need in the lab, and basic molecular biology techniques, since some team members had no previous research experience.

Concise Guidelines for iGEM Researchers

(See above in Researcher Safety for a more detailed description of endocrine disruptor hazards)
• When handling cells, DNA or chemicals, researchers are required to wear gloves and change them often. This protects us and our experiments.
• Gel imaging is still done under UV, so we minimize researcher exposure by:
  • Making sure the image box is closed before turning the UV light on, and making sure the UV light is off before opening it.
  • Performing gel extractions with a blue light box, which only requires amber glasses

   

• Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

   

  Ideas for General iGEM Safety

  We believe requiring more documentation of submitted parts will help improve the safety of iGEM teams. Having more information about what we are working with allows us to design better experiments, understand interactions between parts and be aware of possible dangers to ourselves or the environment caused by the BioBrick. Some ways we think teams can help make their documentation even better:

  • Cite literature used in the creation of the BioBrick
  • Provide documentation of the creation and use of the part by writing directly on the Part page or linking to the team's notebook or project page
  • If possible, sequence the part right before submitting it to the Parts Registry. Use that sequence rather than the intended or planned sequence in the part specification. If it is different from the intended sequence, note that on the Part Design or the BioBrick's main page.
  • Fill out the experience page with data showing how the part worked for a specific test or link to the team wiki about use of that part. Experience pages are useful for helping teams decide what part to use if there are multiple choices to fulfill their need for their project.