Team:NTNU Trondheim/Safety

From 2012.igem.org

NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Safety It's what comes first

Contents

Question 1


Question

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

Answer

In this project two organisms were used, following are the two organisms listed with biosafety level.

Organism Biosafety
Escherichia Coli Level 1
Corynebacterium glutamicum Level 1

Before starting to work in the lab, a risk assessment (developed by the HSE group) was done of all the equipment and procedures to be used in this project. It had to be approved by the HSE group.

The grading system used 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. The risk assessment for the project is given below. For the probability of unwanted incident, the grading system ranges from 1 to 5, where 1 is very low probability (once every 50 years, or less), 2 is low probability (once every 10 years or less), 3 is moderate probability (once a year, or less), 4 is high probability (once a month or more often) and 5 is very high probability (happens every week).

Activity Possible unwanted incident/strain Probability assessment (1-5) Consequence, human being (A-E) Consequence, external enviroment (A-E) Consequence, eko/material (A-E) Consequence, reputation

(A-E)

Risk value Comments/status/ Improvement suggestions
Agarose gel electophoresis Long term health risk from GelRed (unknown effect) 1 Unknown A A B Unknown 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.
Autoclave Rapid pressure fall due to opening the autoclave to soon my cause hot liquid burns on eye or skin 2 C A A B 2C Will not happen if instructions are followed
Bacteria class 1 and recombinant bacteria Release of GMO to environment. Bacterial infections. 2 A A A B 2A
Bunsen burners Skin burns 3 B A A A 3B New rules how to handle burners+ ethanol baths already installed.
Bunsen burners Initiation of fire 3 C A B A 3C New rules how to handle burners + ethanol baths already installed.
General lab work Deviation from established safety rules and routines -- -- -- -- -- -- 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 Exposure for dangerous for health chemicals: Phenol-chlorophorm mix 2 C A A B 2C Phenol-chloroform mix requires the work in a ventilation hood only. All waste should be placed in a special box for hazardous materials.
Environmental samples for cultivation and DNA isolation, preparation of Bacterial infections, production of toxic/infectous compunds from cloned DNA. Usage of hazardous compounds (e.g. chloroform) for DNA isolation. 1 Unknown A A C 1 - Unknown In general mild injuries, that can be avoided by present rutines and senseable carefulness during lab-work.
Dry ice/etanol Eye injury. Frostbite 1 B A A B 1B Eye-protection and gloves for handling the dry ice
Luminometer and fluorometer, use of Possible exposure health injurious chemicals. 2 A A A A 2A See MSDS for relevant chemicals
PCR Exposure to DMSO when used 1 A A A A 1A PCR-machine should be in ventilation hood when DMSO is added, use lab coat and gloves
pH-adjustments Exposure to corrosive chemicals: Skin and eye damage 2 B A A B 2B Eye-protection and lab coat
Sentrifugation (Sorvall + table) Damage caused by loose rotor 1 C A B C 1C
Supercompetent or electrocompetent cells, making of Exposure of skin and eyes to extremely cold liquid (nitrogen or ethanol) 2 C A A B 2C Conducted with extreme caution, and using eye-protection and gloves.
Ventilation hoods, use of Exposure to hazardous chemicals due to unsufficient airflow (effects on local hood or other hoods) 1 C A A B 1C Can be prevented by maintaining sufficient airflow
Waterbaths and shaking waterbaths, (heat and water quality) Electrical hazards, Burns 1 B B A A 1B Regular service by trained personnel. Heat-proof gloves must be used during handling of hot items.

Hot surfaces must be signed.

All of the participants have had previous lab courses. In these courses the participants have learnt about use of safety equipment, first aid, fire extinguishing, proper dispose of waste, and reading and understanding of safety data sheets of the substances in use. Use of different kinds of equipment and methods used have been taught, as well as writing scientific reports. The labs in use in this project are all approved for working with gene modified organisms (GMOs).

Lab coats, safety glasses and disposable gloves are used when necessary and when required by the HSE guidelines and rules. Materials such as restriction enzymes, buffers, PCR and Gel Purification kits are all bought from biotechnology companies. There are none additional safety precautions to be made than the ones already described. When making solutions of antibiotics, gloves and a fume hood are used, in addition to lab coat and glasses.

When the lab’s not in use, it is locked with all of the equipment and materials/organisms safely stored away. A person that doesn’t belong there will not be able to enter. There are also security personel at the school at all times.

Since these bacteria have plasmids with antibiotic resistance genes for selection, these can be harmful if released into the environment. To be sure, never bring GMOs outside of the lab and all of the materials and equipment contaminated by the GMOs, will be kept in special containers to be autoclaved. It any of the organisms were released to the environment, the biggest problem would be if the antibiotics resistance genes were transferred to other organisms. The natural occurring organisms can then influence humans, plants and animals. As long as the rules and regulations mentioned above (and also the rules and regulations give by the government, university and institute) are followed, this will not be an issue. The rules and regulations of the government, university and institute are given in the answer to question 3.

Question 2


Question

Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes,

Answer

In this project the goal is to get E. coli to react to specific environmental factors that are found around cancer cells, such as high lactate and low oxygen concentrations. These qualities are not connected to the toxicity, infectivity or pathogenicity of the organism and are therefore considered not harmful. The strain of E. coli used is also not pathogenic (the DH5ɑ strain).

There is also one gene expressed under a constitutive promoter. The protein expressed named Colicin is a bacteriocin, a toxic for bacteria, and is produced by E. coli. Because it's produced in E. coli, it's a toxin humans are exposed all the time, through their intestines. It is then not considered harmful. Some scientists even consider using it as an anti-Listerial agent in meat products. ([1], 05.09.12)

Question 3


Question

Is there a local biosafety group, committee, or review board at your institution?

Answer

The Health, Security and Environment (HSE) group at the university has certain rules and regulations used in all laboratories. These are rules of work environment and safety, information about these can be found here: http://www.ntnu.edu/hse. Lab work regulations can be found in the HSE handbook: http://www.ntnu.edu/hse/labhandbook. All of the rules and regulations are according to the rules set by the Norwegian government and the Norwegian Biotechnology Advisory Board (http://www.bion.no/english/norwegian-regulation). The project is not in conflict with any of the rules and regulations from the government, university and institute.

Recombinant gene technology is also regulated in Norway by Lov om framstilling og bruk av genmodifiserte organismer m.m. (genteknologiloven) [Law on the production and use of genetically modified organisms, link in norwegian] and Forskrift om innesluttet bruk av genmodifiserte mikroorganismer (Regulation on the contained use of genetically modified microorganisms, link in norwegian)

In addition, all work at NTNU is regulated by the norwegian Working Environment Act (arbeidsmiljøloven, link in norwegian) and internal regulations. Of particular importance at the Department of Biotechnology is the section on chemical and biological hazards (§4-5). Other governmental regulations also apply.

Question 4


Question

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?

Answer

In synthetic biology today, antibiotics are used as means for selecting the cells with the right plasmid. But, as mentioned, this can be a risk to other organisms if released into the wild. It it would be possible to select the GMOs with the right plasmid on basis of something else, it could make the risks of GMOs lower.

If it is possible, a good thing would be to make the organisms used in the lab unable to live in the wild. If they need very specific conditions to survive, the probability of them surviving in the wild or doing any other damage, will be lower.



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