Safety Protocols

Experiments were conducted in the Undergraduate lab of the Dept. Of Biotechnology at IIT Madras. The lab is confirmed to the standards for a bio-safety 1 lab according to the Center for Disease Control and Prevention,Atlanta. 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. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by appropriate risk assessment techniques. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.

Contents 1 Standard Microbiological Practices 2 Safety Equipment (Primary Barriers) 3 Laboratory Facilities (Secondary Barriers) 4 Simple Questions

Standard Microbiological Practices

1. 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.
2. 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.
3. People wash their hands after handling viable materials, after removing gloves, and before leaving the laboratory.
4. 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.
5. Mouth pipetting is prohibited .Instead mechanical pipetting devices are to be used.
6. Policies for the safe handling of sharp instruments are clearly instructed.
7. All procedures are performed carefully to minimize the creation of splashes or aerosols.
8. Work surfaces are decontaminated atleast once a day and after any spill of viable material.
9. 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)

1. 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.
2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
3. 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.
4. Protective eyewear should be worn while conducting experiments in which splashes of microorganisms or other hazardous materials are anticipated.

Laboratory Facilities (Secondary Barriers)

1. Laboratory has doors for access control.
2. A sink is provided in the Laboratory for washing hands.
3. The Laboratory is designed such that it can be easily cleaned.
4. 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.
5. Laboratory furniture is capable of supporting anticipated loadings and uses. Spaces between benches, cabinets, and equipments are accessible for cleaning.
6. It is made sure that the Laboratory is cut-off from the external environment.
7. We have an accesible compartment in the lab for MSDS(Materials Safety Data Sheet) where in we have a roster of all the chemicals and what needs to be done in case of accident.

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

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

Researcher Safety

• Chassis: E.coli is the most commonly used gram-negative bacterial chassis in Molecular Biology.
  They are normal flora of the human gut, and can benefit their hosts by producing Vitamin K2,and by preventing the establishment of other pathogenic bacteria within the intestine. Some strains of E.coli are also pathogenic, associated with sever diarrhea.
  However the strains of E.coli used in lab are known to be non-pathogenic. However researchers are advised to use standard laboratory safety equipment and procedures while handling the cultures including wearing Lab coats, Safety Glasses etc.
• EtBr: Ethidium bromide is an intercalating agent and is/was commonly used as a fluorescent tag (nucleic acid stain) for agarose gel electrophoresis in our lab. Ethidium bromide is thought to act as a mutagen because it intercalates double stranded DNA (i.e. inserts itself between the strands), deforming the DNA. This could affect DNA biological processes, like DNA replication and transcription. Hence, we do not wash the gel in EtBr solution. Instead, EtBr is added in minimal concentration to the agar solution before it solidifies. It is strictly observed that gloves are used when handling EtBr or EtBr-containing gel. The gels are discarded considering them to be 'Hazardous Waste'.
• Sodium Azide: Sodium Azide is a strong respiratory toxin which inhibits the activity of cytochrome oxidase and acts as a bacteriostat. It is however also highly toxic to mammals. Researchers are required to be extremely careful while handling sodium azide solutions and cultures with sodium azide. Lab Coats and Gloves are a must, they must also be informed about the hazards and the first aid procedures in case of azide spills.

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

Yes, our project is supported by the Dept. of Biotechnology, IIT Madras. They have provided us with Biosafety level 1 facilities for all our experimental work.

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 proteorhodopsin construct as a tool for selecting transformed mutants. We have designed a new plasmid, pSB1Pc(BBa_K572100) that does not contain antibiotic resistant gene. Instead it has that will aid the cell growth and provide a selective pressure in a limited nutrient media. We envisage that this could make antibiotic based screening technique obsolete and could mitigate the risks attached with Horizontal Gene Transfer of antibiotic resistance genes.
3. Our protein expression plasmid, pSB1Pe(BBa_K572200) is a plasmid sans antibiotic resistance gene but uses proteorhodopsin for selectivity. As our system works in a low carbon media, we have provided carbon stress promoter, PcstA(BBa_K118011) to increase production in this condition. Hence this construct provides higher growth with proteorhodopsin and production in carbon stress.

References

• Cupillard, L. et al., 2005. Impact of plasmid supercoiling on the efficacy of a rabies DNA vaccines to protect cats. Vaccine 23: 1910-1916.
• JM Tiedje et al., 1989. The planned introduction of genetically engineered organisms: Ecological considerations and recommendations Ecology, 70(2), 1989, pp. 298-315
• Efstathia V. Scoulica. et al., Spread of blaVIM-1-producing e. coli in a university hospital in Greece. Genetic analysis of the integron carrying the blaVIM-1 metallo-β-lactamase gene,Diagnostic Microbiology and Infectious Disease Volume 48, Issue 3, March 2004, Pages 167-172
• Mae-Wan Ho, ISIS Report - May 4, 2001., Horizontal Gene Transfer Happens - II