"

From 2011.igem.org

Protocols

Below, please find the steps that we followed to carry out molecular cloning, create recombinant DNA parts, and construct microfluidic channels.

Molecular Cloning Protocols

PCR Reaction

Note: Keep everything on ice and add all volumes in a PCR tube.

Public Safety
It is a common misconception that bacterial strains used in research are dangerous to public health and safety. However, the bacteria we used, DH5a electrocompetent E. coli, are non-pathogenic and unlikely to survive outside of a laboratory setting. To insure minimal risk to public safety, bacteria were killed with 10% bleach before disposal. We perceive no potential threat to public safety from our project. The biochemical pathway we used converts tryptophan, a common amino acid found in many foods, to prodeoxyviolacein, a green pigment. Both chemicals and all intermediates are non-toxic and should present no threat to public health or safety. Furthermore, no gloves were allowed to leave the laboratory, in order to prevent contamination of pubic areas.

Environmental Safety
Our project poses no identifiable threat to environmental safety. As stated above, the DH5a E. coli used in our project is not able to survive outside the lab, and all cells disposed of safely after disinfection with 10% bleach. Bio-hazardous and flammable chemicals were disposed of by following the proper regulations, as stated above. As stated above, no gloves were allowed to leave the laboratory so that chemical and biological hazards were restricted to the lab.

Security Risk
We perceive no threat to security from our project. The final product of our biochemical pathway is a harmless pigment which could not be used with malicious intent. While our "biofactory" could be adapted for use in the synthesis of a harmful chemical, the microfluidic device is simply tool to cut costs of synthesis in an industry setting, and does not present any new security threat despite its potential to be misused.

Safety of BioBricks
None of the BioBricks submitted to the registry raised any safety concerns. The parts which we worked with are GFP, RFP, VioA, VioB, and VioE attached to avitags. These BioBricks already exist in the registry, and the addition of the avitag, which allows the molecules to be tethered to our microfluidic device, does not raise safety issues in the use of these parts. Our last BioBrick, the light lysis package, causes cells to lyse in the presence of green light. This should not cause any safety concerns.

Microfluidics Protocols

Biosafety Rules and Procedures
We complied with Weill hall’s safety requirements in gaining access to lab space, as well as in use of the lab. All safety information and procedures are linked on the main Weill safety page [http://blogs.cornell.edu/whfs/weill-hall-safety-links-and-information/ here].

Weill Hall Safety Personnel
Scott D. Emr is the director of Weill Hall which is the building our lab is located. There is also a Weill Hall Safety Committee. While we did not personally meet as a group with either groups, we talked Dr. Archer who is in charge of our particular lab space. She approved of our project and helped us with the safe construction of our microfluidic mask.

Safety Training We received safety training from two online courses that all members were required to pass in order to gain access to the building. These were Lab Safety and Chemical Waste Disposal. Also we received training from our lab instructor on basic safety issues such as waste disposal, use of the fuse hood, etc.