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Team:NTNU Trondheim/Safety
From 2011.igem.org
Safety
The university has a health, safety and environment (HSE) group with set guidelines and rules to regulate the work environment and safety. General information about this can be found at http://www.ntnu.edu/hse. Details on the HSE regulations for lab work can be found at http://www.ntnu.edu/hse/labhandbook. These are all based on the national biosafety regulations and guidelines set by the Norwegian Biotechnology Advisory Board (http://www.bion.no/english/norwegian-regulation) and the acts set by the government.
Lab work
The institute for Biotechnology has set some local guidelines and rules that apply for the specific labs that we use in our lab work for this project. The labs used are approved for work with gene-modified organisms (GMOs). Prior to the commencement of work in any of these labs, every person is given the essential training. The training covers the statutory regulations and safety provisions, the use of equipment and protective equipment, fire extinguishing, first aid, use of the chemical substances index and safety data sheets, proper waste disposal and reporting. The wetlab part of the NTNU iGEM team has all attended this training and is following the set lab guidelines and rules in their work.
We of course follow general lab safety and wear lab coats, disposable gloves and safety glasses when required by the safety guidelines and rules. The materials used in this project are mostly standard kits, restriction enzymes and buffers that are bought and come pre-made from biotechnology companies, and does not require any other safety precautions than the general ones already described above. This also goes for the preparation of solutions like growth medium and gels for gel electrophoresis. When doing gel electrophoresis we use GelRed instead of ethidium bromide, as it is said not to penetrate cell membranes and thus should not be able to work as a mutagen. However, to be on the safe side, we always wear nitrile gloves when working with GelRed.
Assessment of our project and BioBricks
Our project does not inflict with any of the regulations, guidelines and acts set by the university, institute and government. None of the bacteria and BioBricks used or made in this project raises any serious safety issues for the public health, environment and security. Our activity and use of biological material corresponds to class 1 in the Norwegian regulation for protection against exposure to biological agents and use of GMOs. Class 1 cover use that does not involve any significant risk for human's and animal's health and environment. This corresponds to WHO risk group 1 in the classification of infective microorganisms.
Our aim in this project is to make bacteria that fluoresce when stressed. This is not a property that is considered harmful or is related to the organism’s pathogenicity, infectivity or toxicity, and the strains of E. coli used (DH5alpha and TOP10) are non-pathogenic to people of normal health. Neither do they carry an F factor (fertility factor) and are therefore unable to perform conjugation. The strains used are optimized for lab use only and should not be able to survive in a foreign environment. Plasmids carrying genes for antibiotic resistance are used for selection, and the bacteria carrying these plasmids could in theory be of health and environmental concern if not handled properly. As a golden rule, GMOs should not under any circumstances be brought outside the lab. To overcome any possible safety issue in relation to the points described above, all biological material and equipment that has been in contact with this biological material are contained in special waste containers within the lab and autoclaved before disposal.
If any of our organisms where to be released to an environment outside the lab, either by design or accident, the biggest threat in theory would be the transferring of genetic material and conferment of antibiotic resistance to other microorganisms in natural habitats and microorganisms related to human, animal or plant diseases. Antibiotic resistance is of huge concern when it comes to the treatment of infectious diseases and the risk of this happening should always be taken seriously and appropriate safety steps should be made. The steps described in this text, together with the set guidelines and rules, are considered sufficient to prevent this.
Risk Assessments
The following risk assessments for the lab procedures we are using have been made by the institute’s HSE group. The grading system for personal and environmental risk assesments ranges from A to E, where A is no/very low risk, B is low risk, C is moderate risk, D is high risk and E is very high risk:
| Activity | Safety Procedures | Personal risk | Env. risk | Comment |
|---|---|---|---|---|
| Agarose gel electrophoresis, (GelRed) | Nitrile gloves, protective eyewear (with UV filter), face shield when needed.
Gelred used for staining | GelRed: Unknown, UV: B | A | Gelred is said not to penetrate cell membranes, and thus should not act as mutagen even if it is DNA-binding. Gloves also minimize the risk for exposure. UV damages on unprotected skins/eyes if instructions not followed |
| Antibiotic-stock solution, make and use of | Gloves, handle powder only inside fume hood | A | B | May cause allergic reactions if instructions not followed. May causlead to multi-resistant bacteria if not disposed correctly. |
| Autoclave | Thermoresistant gloves
Eyeprotection lab, Instructions posted for not opening autoclave too early and for not overfilling bottles or closing their lids completely. | C | A | Rapid pressure fall due to opening the autoclave to soon may cause hot liquid burns on eye or skin. Will not happen if instructions are followed. |
| Bacteria class 1 and recombinant bacteria | Autoclave accessible. Inactivation of genmanipulated bacteria in contaminated material and waste. Labcoat mandatory. Lab bench surfaces resistant to water, acid, alkali, solvents, disinfective agants, decontaminating agents and easy to clean. Transport between labs only in closed containers. Good microbiological practice. | A | A | Risk include Release of GMO to environment, bacterial infections. Low because DH5 alpha are crippled. |
| Use of open flames – (e.g. sterilization with bunsen burners) | Bunsen burner must not be left burning | B | A | Risks include skin burns and fire. New rules on handling installed. |
| General lab work | Safety rules according to risk assessment | - | - | No injuries requiring more than simple first aid in these laboratories for the past 5 years, the present rutines seem sufficient |
| DNA/RNA isolation and purification | Use gloves and eyeprotection during steps including NaOH. Use fume hoods for procedures containing chloroform, phenol or if it is indicated in the kit manual | C | A | Phenol-chloroform mix requires the work in a ventilation hood only. All waste should be placed in a special box for hazardous materials. |
| Preparation of medias for growing bacteria | According to MSDSs of relevant chemicals | - | - | Depends on the chemicals |
| PCR | Fume hood when DMSO is added | A | A | PCR-machine should be in ventilation hood when DMSO is added, use lab coat and gloves |
| pH-adjustments | Nitril gloves, eyewear, shoes/shoe bags with protection agains acid/base spill, sufficient ventilation (hood/”cap”) | B | A | Eye-protection and lab coat |
| Sentrifugation (Sorvall + table) | Accurate balancing, accurate attachment of rotor, not exceed maximal G-forces fore each type of tube | C | A | Danger is damage caused by loose rotor |
| Supercompetent or electrocompetent cells, making of | Eye protection, gloves and protective shoes/shoe bags when handling liquid nitrogen or ethanol/dry ice bath. Shoes must be easy to take off in case of spill into (never use rubber boots) | C | A | Conducted with extreme caution, and using eye-protection and gloves. |
| Ventilation hoods, use of | Opening minimized when not in use, correct settings when in used for protection | C | A | Exposure to hazardous chemicals due to unsufficient airflow (effects on local hood or other hoods). Can be prevented by maintaining sufficient airflow |
