Team:Tec-Monterrey/ITESM14 safety protocols.html

From 2014.igem.org

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Within our institution is self-regulated and trains its researchers with biosafety guidelines without a formal online version of them, in order to work in the lab many of us have already received a basic training in charge of the main laboratory. The project needed approval from the equivalent group of an Institutional Biosafety Committee at our institution which is formed by Dr. Silverio García Lara, Dr. Rocio Diaz and Dr. Guy A. Cardineau, who is one of the advisors of the team. They gave us feedback regarding the mammalian cell lines and the bacteriophage that were going to be used, which are some of the most sensitive elements of our project. Thus, actions were taken at different levels to protect the people within the lab and other people who also use it because our job is to minimize the probability of being exposed to any danger.

Risks to the people working in the lab

About the bacteriophages, we are needed to be extra careful when working because it has a peptide that enables the internalization when it finds the cancer-specific receptor (GRP78). This receptor is found in cells that have stress in the endoplasmic reticulum, caused by hypoxia and low glucose levels in the environment (tumor-like environment). According to various studies the amount of receptors in the normal cells is not enough to make probable the internalization. So the risk of handling the bacteriophages in controlled areas is very low.

Risks to the safety and health of the general public

We desingned our bacteria to produce its recombinant bacteriophage under a quorum sensing system and also, it will only transfect cancer cell lines, which limits and lowers the possible risks. Also the strains that we are going to use cannot survive outside laboratory conditions for more than two weeks as they are derivated from E.coli K12, so the probability of producing bacteriophages outside the lab conditions is very low.

Environment and waste disposal

To reduce these kind of risks we are going to work according to the safe lab practices and we are going to dispose the wastes correctly. In addition, we are using E. coli strains that cannot survive outside laboratory conditions for more than two weeks. So the risk of an accidental production of our therapy outside lab conditions is limited.

We summarized some of the most important safety measures in each protocol, in the following table.

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Protocol	Hazardous Materials	Safety Measures	Type of hazard
Miniprep (plasmidic DNA extraction)(003)	Acetic acid EDTA NaAC NaOH HCl	The solutions with EDTA were made with nitrile gloves to prevent any possible contact with the skin, as it is known that EDTA has suspected effects on the reproductive system. Solutions using NaOH and NaAc were made in a laminar flow hood to prevent inhalation.	Environmental Individual Lab. Personnel
Agarose gel DNA electroforesis and purification (007)	Ethidium Bromide UV light	Ethidium Bromide intercalates into the DNA double strand, as a strong intercalator is mutagenic and carcinogen. Inside the lab there is a special workplace where the number of people who can use it is restricted. To keep the biosafety all of the recommendations were followed, and we used nitrile gloves that are less permeable to EtBr than latex. There was a special disposal bin for all EtBr–related residues. DNA can be extracted from the gel; thus, protection is used to avoid the contact with the UV light.	Lab. Personnel Environmental Individual
Restriction enzyme analysis(006)	Restriction enzymes	To protect the user, gloves must be worn at all times.	Environmental
Escherichia coli Transformation by CaCl₂(001, 002)	Chlorine Non-transformed E. coli	E. coli non-transformed strains should not escape, so we perform every transformation under a biosecurity chamber. Any residues generated in this protocol that had contact with biological material are then disposed in a biohazard bag that is sealed and autoclaved in 121°C and 15 psi for 15 minutes.	Environmental General Public
Escherichia coli Transformation by Electroporation (004, 005)	Non-transformed E. coli	E. coli non-transformed strains should not escape, so we perform every transformation under a biosecurity chamber. Any residues generated in this protocol that had contact with biological material are then disposed in a biohazard bag that is sealed and autoclaved in 121°C and 15 psi for 15 minutes.	Environmental General Public
SDS-PAGE & Western Blot	Acrylamide ATEMED PSA SDS Coomassie Blue ß-Mercaptoethanol	The polyacrylamide gel is done by adding TEMED and PSA to Acrylamide, which are harmful for the respiratory tract, so for its use we need to have precaution. Acrylamide is known for being a strong neurotoxin, so nitrile gloves are used. </br> In the sample preparation, ß-mercaptoethanol and SDS are needed, and both have toxic effects, especially ß-mercaptoethanol. To keep the biosafety standards, it was necessary to minimize the time of exposure to any of these materials, by using nitrile gloves and within the designated space. </br> Coomassie Blue is a known flammable toxin that targets some of our organs. It is prepared in a “fixating solution”, which contains methanol, acetic acid and water. For the preparation of this solution, an air-extraction cabinet is needed. The dispose of all the generated wastes in a bin we labeled “PAGE wastes”, separated from the normal wastes.	Individual Lab. Personnel Environmental General Public
Cell lysis and Protein Extraction(006)	Imidazole Tween® 20	Tween® 20 works as a detergent, and can be very harmful to the skin. Imidazole has this same problem, when this protocol is made gloves must be worn at all times. Eye protection is recommended as well, but it is not essential. In case of contact, the surface must be thoroughly washed.	Individual Environmental
Red Lambda Protocol (009)	IPTG MOPS	Some chemical reagents used with this system are dangerous if handled carelessly. Take care when using chemical reagents, besides the previously mentioned for gel electrophoresis. MOPS are salts that can be very harmful to the eyes, while IPTG is harmful by inhalation, in contact with skin and if swallowed. As a result, gloves must be worn at all times.	Individual Environmental Lab. Personnel
Quorum sensing assay (008)	PBS	This is a very harmless protocol, just gloves must be worn at all times in case the salts that are in the solutions are spilled.	Individual
Mammalian cells apoptosis assay (016)	7-AAD	7-AAD is a potential carcinogen. It is recommended that the user wear protective clothing, gloves, and eye/face protection in order to avoid contact with skin and eyes. the disposal is done in the proper chemical disposal.	Individual Lab. Personnel General Public
LAL assay(014)	E. coli Endotoxin	It is probably one of the safest ways to determine the toxicity of the proteins. The presence of the endotoxin is the principal safety risk in this matter.	Individual General Public
PCR mutagenesis of phage (015)		Personal protection and gloves must be worn at all times. The probes must be designed to avoid any mistake in the mutations that could be dangerous. If your PCR uses microwaves to heat, you must be careful as well.	Individual Environmental
Phage purification(011)	Ammonium sulfate	Mutated phages with pep42 peptide will be able to internalize to mammal cells. Although their action is localized, it is wise to be careful with them. Lysol must be applied wherever the phages are being used; also, all the procedures must be carried out in the laminar flow hood, and personal protection as gloves and coat must be worn at all times. </br> Until conclusive tests are done, this precautions must be taken. </br> Ammonium sulfate is a health hazard in contact with skin and if inhaled. Thus, gloves must be worn at all times, and care must be taken when the solutions for precipitation are made.	Individual Lab. Personnel Environmental General Public
Phage quantification(012)	Biuret reagent	All precautions mentioned above must be considered. The cells for the spectrophotometer must be cleaned to avoid contamination with other cultures, and people. Biuret reagent relatively very hazardous because it has Cu. It also poses a notable environmental threat, so it was deposited in the dangerous chemical residues bin of the lab.	Individual Lab. Personnel Environmental

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