Device Designs

Hef1a:Gal4-VP16, UAS:Reporter

This circuit represents the behavior of the Gal4 activator. Hef1a is a constitutive promoter that will express Gal4-VP16 that can later bind to a UAS promoter, leading to the expression of downstream reporter genes.

Hef1a:LacI, Hef1a-LacOid:eYFP

Here we represent the activity of the LacI inhibitor. LacI binds to the upstream LacO site and can inhibit expression of downstream reporter genes.

Hef1a:Gal4-VP16, UAS:LacI, Hef1a-LacOid:eYFP

Here we see a circuit that integrates the two systems described above. By tying LacI expression to the Gal4/UAS system, we can activate expression of lacI by GV16 and thus inhibit expression of a reporter gene. This results in overall repression of reporter expression.

Gal4-VP16, UAS_NCad-2A-eYFP

Here we created a part that is activated by the Gal4-VP16 transactivator to produce N-Cadherin joined to eYFP by a 2A tag, which is cleaved to result in the expression of both proteins.

Hef1a:rtTA3, TRE:Mnt-VP16, minCMV-4xMnt:mKate

Here we represent a circuit that has an upstream rtTA3 system feeding into the Mnt activator system. When bound by doxycycline, rtTA3 is capable of inducing expression at the TRE promoter. This in turn leads to expression of the activator Mnt-VP16 which can bind to Mnt sites and activate expression of the reporter gene (mKate, a red fluorescent protein, in this case).

Hef1a:rtTA3, TRE:CI434-VP16, minCMV-1xCI434:mKate

This circuit is very similar in design to the above, instead using a different orthogonal activator-promoter pair: CI434. In this schematic, doxycycline induced expression of CI434 leads to activation of reporter expression downstream of the CI434 binding site (minCMV-1xCI434).

Safety

Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety?

This summer, our team worked on cell patterning in mammalian cells. Part of our team worked with E. Coli in a BL1 lab, and a smaller group worked with mammalian cells in a BL2 lab. Both groups within the team adhered to national and local safety protocols. Extra care was taken not to cross-contaminate lab spaces. Cross contamination from these settings was minimized by designating specific equipment for mammalian cells and for bacteria, as well as immediately changing personal protective equipment when moving between the different lab spaces.

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 bacteria and our mammalian cells do not contain any BioBrick parts that code for hazardous proteins or molecules. We also determined that none of our circuits would survive if released outside the lab and thus, the circuits pose no safety concerns to researchers, the public, or the environment.

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?

The EHS (Environment, Health, and Safety) Office is MIT's biosafety group that enforces lab safety in all labs on campus. They provide safety training, waste management services, and resources for safe lab practices. The safety training included General Biosafety for Researchers, Managing Hazardous Waste, General Chemical Hygiene, lab-specific training, and Bloodborne Pathogen Training. All undergraduates were trained by the EHS to work safely in BL1 labs, and students working with mammalian cells received BL2 lab safety training. EHS is an integral part of our biosafety system and continues to be involved by conducting daily lab safety inspections.

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?

Our MammoBlock construction standard not only aids in the construction of mammalian parts but also facilitates safer and easier storage of these parts. The MammoBlock standard uses bacterial entry vectors which allows mammalian parts to be stored in BL1 conditions.

Our team is greatly concerned with the safety issues regarding future iGEM research, especially relating to mammalian cells. This standard has allowed us to enter mammalian parts into the registry, and will facilitate the safe shipping and submission of mammalian parts constructed by future iGEM teams. and competitions.