Safety

Pretty much everything in a lab can be a risk if not handled properly. Dangerous chemicals, equipment and microorganisms all pose a risk to human health if not handled and disposed of correctly. Safety in and outside of the lab is something we as a team valued very highly and here follows a description on how we took measures to make our lab environment as safe as possible.

General lab safety

Almost everyone in our team has lab experience and has gone through the safety procedures multiple times and also have general knowledge on what is known as “Good laboratory practice”. This didn’t stop us from having a thorough safety instruction at the beginning of this year’s iGEM. This meant reviewing proper waste disposal, fire safety and emergency exits, biosafety, proper handling of chemicals and materials and last but not least, handling bacterium and other microorganisms and how to protect yourself and others while working with them.

The general lab safety was an ongoing theme throughout the summer as we handled dangerous chemicals as we went along. When a new chemical or the like was introduced the safety measures on how to handle it was addressed as well.

Dangers when working with microorganisms

In our lab we were only allowed to work with bacterium with safety class 1. If we needed to work with any organism with a higher safety class we had to take our work elsewhere. Any organism we took into the lab had to be cleared by prefect of the faculty.

The bacterium we mainly used in this project was the Escherichia coli DH5α. This particular organism is a class 1 bacterium and non-pathogenic to humans. We still took safety precautions when handling the DH5α, such as always wearing a labcoat when in the lab and using gloves and goggles when they were needed. This also included sterilizing the work benches with 70% ethanol both before and after every procedure to avoid growth and further contamination. Although the harmful effects to humans is limited with this particular strain of E.coli you still run a risk of spreading antibiotic-resistance from the plasmids used in iGEM to other bacterium if you don’t take these safety precautions. Therefore all waste that contained bacteria was treated with jodopax, an iodine based disinfectant, before being discarded.

Apart from the E.coli DH5α another strain of E.coli were used. This strain, the BL21-DE3, is also safety class 1 and the same precautions were taken as for the DH5α.

Other organisms

The pathway responsible for breaking down naphthalene was extracted from the Pseudomonas putida PpG7. This organism is safety class 1, but since there have been some cases where this bacteria have infected humans we took extra safety precautions when working with it; it was handled and kept in a fume hood. Since this bacteria consumed naphthalene as a carbon source it was always handled with gloves and goggles in a fume hood when naphthalene crystals had been added.

Dangerous chemicals

• Acrylamide solution-This compound was used in the SDS-PAGE. It’s poisonous and can cause organ damage after long exposure. It can cause irritation to the skin and eyes and is also harmful when inhaled. On top of all this it’s also linked to causing genetic damage and cancer. Because of all this the compound was always handled in a fume hood. Thick nitrile gloves was used along with goggles. The one working with the substance was also always extra cautious when handling this chemical due to it’s dangerous properties.

• Ammonium persulfate-This inorganic compound is used as a source of free radicals in the SDS-page procedure. It’s a strong oxidizing agent and can intensify fire. It’s harmful when ingested and can cause irritation to the airways, skin and eyes.

• CTAB-Cetyl-trimethylammoniumbromide. CTAB is an amine based cationic quaternary surfactant. This compound contains bromide and therefore should be handled with care. According to the MSDS from Sigma-Aldrich it can cause irritation when it comes in contact with skin and because of that it was always handled with gloves and goggles and since inhalation can cause damages to the lungs it was always handled in a fume hood. When the plates containing CTAB were to be disposed of, a special waste bucket was used. When the chemical was not used it was kept in a safety locker.

• Hydrochloric acid-This is a common compound in any lab. When diluted it’s something that you have to be careful with, but in it’s concentrated form you have to be extra careful. It was always kept in a special cupboard for corrosive chemicals and thick gloves and goggles was always used when handling it. It was also always handled in a fume hood.

• Metal salts-Pseudomonas minimal medium (PMM) is a medium specifically designed for growing Pseudomonas strains. To this media a number of metal salts are added as a source for trace elements. Among these salts were CuSO₄, FeSO₄, MnSO₄ and Co(NO₃)₂. All of these salts used were considered harmful when ingested and could have a negative environmental impact. Because of these all of the medium that contained metal salts were disposed of in a separate waste container and handled properly.

• Naphthalene- Naphthalene is an organic compound made up of two aromatic rings. Since this compound is flammable it was always kept in a fume hood when used. When not used it was always kept in the flameproof locker. Naphthalene is harmful when consumed and is suspected to cause cancer and was always handled with gloves and caution. Since it’s very harmful to aquatic organisms it was disposed of in a separate container and taken care of by the university.

• Orcinol monohydrate-This is a natural phenolic organic compound. It can be harmful when ingested, irritating upon skin contact, causes serious irritation to the eyes and causes irritation to the airways. It was always handled in a fume hood with gloves and goggles. It is both light and air sensitive and should be kept in an inert gas. It was also disposed of in a special waste.

• Phenantrene-This polycyclic aromatic hydrocarbon is composed of three benzene rings. It is formed by incomplete combustion and can thus be found in cigarette smoke and the fumes from industry. In its pure form it’s a white powder that has blue fluorescence. It is harmful when ingested and can cause irritation when it comes to contact with skin. It also causes irritation when inhaled and causes severe irritation of the eyes. It is also very harmful to the environment and can cause long term damage to aquatic organisms. Although many PAHs have been linked to a higher risk of cancer, not enough information about phenantrene exists to link it specifically to cancer.

  Because of all this it was always kept and handled in a fume hood. When handled one always used gloves and safety goggles. It was disposed of in separate container and then taken care of by appointed personnel at Uppsala university.

• SDS- Sodium dodecyl sulfate. This compound is used in the SDS-PAGE procedure to denature proteins. It’s a solid compound that is highly flammable and was therefore always kept in a locker designed to keep flammable compounds. It’s harmful when ingested and due to its corrosiveness it causes irritation to the skin and eyes. It also causes irritation to the airways. It was always handled in a fume hood with nitrile gloves and goggles.

• Sodium salicylate- This compound is a salt that consists of sodium ions and salicylic acid. It can be harmful when consumed and can cause serious irritation to the eyes and skin and it was always handled with gloves and goggles. When diluted it was kept in a cold room on a specific shelf.

• Sulfuric acid-This is considered a strong acid and it is highly corrosive. It was kept in a locked cupboard when not used and always handled in a fume hood. Since adding water to this acid causes heat generation which causes the water to boil and to avoid injuries from splashing we always used goggles and thick gloves when using this acid.

• TEMED-N,N,N′,N′-Tetramethylethylenediamine. This compound is used in the SDS-page procedure to stabilize the free radicals. This compound comes as a liquid and is very flammable. It was therefore always stored in a locker designed to hold flammable compounds. When handled it was always kept in a fume hood. It’s also corrosive and causes damage to the skin so nitrile gloves was used. It’s also harmful when ingested and inhaled.

• Triton-X100- This is a nonionic surfactant that has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon hydrophobic group. It is hazardous when ingested and causes serious irritation the eyes and skin. It was always kept in a separate locker and always handled in a fume hood. Since it it’s hazardous for aquatic organisms it was disposed of in the correct way.