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From 2012.igem.org

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Safety Protocols

Experiments were conducted in the Undergraduate lab of the Dept. Of Biotechnology at Jaypee University Of Information Technology. The lab is confirmed to the standards for a bio-safety 1 lab according to the Center for Disease Control and Prevention. Bio-safety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment.

Standard Microbiological Practices
a. Statement of Purpose (SOP) have been developed and are kept for easy access which enlists all the safety measures each experiment should be abided for.
b. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.
c. People wash their hands after handling viable materials, after removing gloves, and before leaving the laboratory.
d. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. People who wear contact lenses in laboratories must wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
e. Mouth pipetting is prohibited .Instead mechanical pipetting devices are to be used.
f. Policies for the safe handling of sharp instruments are clearly instructed.
g. All procedures are performed carefully to minimize the creation of splashes or aerosols.
h. Work surfaces are decontaminated atleast once a day and after any spill of viable material.
i. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method for example Autoclaving. Materials to be decontaminated outside the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. These materials are packaged in accordance with applicable local, state, and federal regulations before removing from the facility.

Safety Equipment (Primary Barriers)
a. Special containment devices or equipment such as a biological safety cabinet is generally not required for manipulations of agents assigned to Bio-safety Level 1.
b. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
c. Gloves are must especially if there is a cut on the hand or if a rash is present. Alternatives to powdered latex gloves should be available.
d. Protective eyewear should be worn while conducting experiments in which splashes of microorganisms or other hazardous materials are anticipated.

Laboratory Facilities (Secondary Barriers)
a. Laboratory has doors for access control.
b. A sink is provided in the Laboratory for washing hands.
c. The Laboratory is designed such that it can be easily cleaned.
d. Bench tops are impervious to water and are resistant to moderate heat, organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.
e. Laboratory furniture is capable of supporting anticipated loadings and uses. Spaces between benches, cabinets, and equipments are accessible for cleaning.
f. It is made sure that the Laboratory is cut-off from the external environment. Would any of your project ideas raise safety issues in terms of:
 researcher safety,
 public safety, or
 environmental safety?

Researcher Safety
Working in a laboratory environment is not without its risks, so we took a variety of steps to ensure the safety of our team during our lab work. We worked in a laboratory environment certified for Biosafety Level 1 work. Advanced synthetic biology research does at times require interaction with potentially dangerous substances. Listed below are those substances which posed the most significant hazards to researcher safety. Again, however, it should be noted that our use of these substances was consistent with Harvard's stringent safety standards. These substances included:
 Ethidium Bromide (EtBr) was used to stain DNA for gel electrophoresis. Although toxic and a suspected mutagen, the harmful effects of ethidium bromide can be avoided by avoiding direct skin contact and inhalation, which can in turn be avoided by proper observance of safety precautions. In order to minimize primary contact, ethidium bromide is directly contacted only with micropipette tips. Skin contact when handling ethidium bromide-stained gels was avoided by the use of nitrile gloves that were promptly discarded for a fresh pair when switching from the task of pouring gels to other lab work. Secondary contact was avoided by the disposal of ethidium bromide-contaminated gloves and micropipette tips, as well as the designation of a specific bench and set of micropipettes exclusively for use with ethidium bromide.

• Ultraviolet (UV) light was used in visualizing stained DNA in gel electrophoresis. To avoid exposure to UV radiation when reading gels, protective, UV-blocking shields were used at all times. When performing gel extractions, exposure was avoided by wearing protective clothing to cover any exposed skin and safety glasses with UV-protection lenses.

• The laboratory strains of E. coli, Methylococcus capsulatus used were non-pathogenic and therefore not a threat to researcher safety. We have conferred various basic antibiotic resistances (including kanamycin resistance, all common features of synthetic biology lab work) to our strains. However, these strains are unlikely to survive in the wild, or in humans specifically—where they would be outcompeted by naturally-occuring bacteria—and thus this resistance does not present a significant problem for researcher safety. In addition, all used materials that contacted the bacteria were decontaminated with 10% bleach, ideally for a 20 minute minimum contact time, before disposal in designated biohazard receptacles.

Public Safety

• All work with live E.coli , Methylococcus capsulatus cells is carried out in the laminar hood that eliminates the possibility of any GMO(Genetically Modified Organisms) from being released out.
• If toxic, flammable, or chemically reactive substances used in our experiments are released, they may post threat to the general public. These include physical hazards or health hazards.
• It is made sure that appropriate measures for waste disposal are taken. Waste is not washed down the sink or thrown into public dust-bins.

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

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?
No. All bio-bricks made are according to the safety guidelines provided by the Center for Disease Control and Prevention.

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, our project safety is governed by the Committee on Bio Safety, Department of Biotech/Bioinformatics, JUIT. We have presented our project proposal to the same committee as mentioned above , and after a review of our materials and procedures, the biosafety office has approved our project and deemed our practices consistent with biosafety regulations.

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?

Antibiotic resistance is a public health problem of increasing magnitude, and finding effective solutions to address this problem is a critical focus of CDC activities.
1. Decreasing the susceptibility of recombinant cells to antibiotics through Antibiotic resistance genes proves to be a high risk to the environment.
2. Our new idea is to use Sac B gene insertion as a tool for selecting transformed mutants. We have designed a new plasmid, that does not contain antibiotic resistant gene. Instead it has Sac B whose expression in the presence of sucrose is lethal to Methylococcus capsulatus. Our results imply that the sacB gene can be used as a positive selection system in Methylococcus capsulatus.
Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible. The link national biosafety regulations in India [http://dbtbiosafety.nic.in/].