Team:Tec-Monterrey/antifreeze/safety

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

We are working in two different projects, thus, in order to assess the risk factor, we consider appropriate to analyze their individual issues in terms of biosafety. First of all, it is important to cover a brief description of both the materials used in the projects, and the organisms employed and their characteristics.

It is important to understand that we are always exposed to risk and no experiment is 100% risk free. Nevertheless, our job is to minimize the probability of being exposed to any danger.

We are working with two different organisms, adding up a total of four E. coli strains and one P. pastoris strain. Using non-pathogenic strains of common use in laboratory as JM109 DE3, BL21 Star, Rosetta Gami and TOP10 F, lowers the risk level the researcher is exposed to.

Regarding the Antifreeze Project, the Escherichia coli strains, all level 1 or 2 of Biosafety (Camacho et.al), are expressing the Antifreeze protein from Rhagium inquisitor, which has no reported toxicity. The experimentation steps in this project consist in simple transformation, inoculation, and bacterial colonies counting, protocols which represent minor biosafety issues.

The antifreeze protein strain could have the ability of surviving freeze-thaw lysis processes, this means that the researcher needs to have further precaution when handling this strain and should use other lysis methods prior to any protein analysis or other non-biosafety chamber needing procedure.

On the other hand, the Allergen Project uses Pichia pastoris, also a non-pathogenic organism on its own (V Balamurugan, 2007). We are modifying P. pastoris cells so that they can express three recombinant proteins with allergenic properties. The allergens produced are Api m 6, Der f 2, and Zea m 14, which are secreted to the medium, increasing the risk of an allergic response in the researcher. Thus, proper care is required when handling these cultures.

Furthermore, the inductors we are using for our promoters (L-Arabinose, IPTG, and Methanol) present no harm to the researcher. Methanol was used with caution as it is known for being a flammable substance.

Also, the experimentation in both projects required us to perform SDS-PAGE technique to detect the heterologous proteins, as neither our allergens nor the RiAFP possess any measurable catalytic activity. Therefore, acrylamide (a dangerous neurotoxin) was used throughout our activities in the lab. As a precautionary measure during the handling of acrylamide, we used nitrile gloves and worked in a specially designated workspace just for the use of this compound. The same previous procedure was followed for the use of Ethidium Bromide during the preparation of agarose gels. We disposed of acrylamide and ethidium bromide in two separate bags, following the biosafety procedures of our institution.

o Public Safety

About public safety, the production of allergens by recombinant organisms can raise biosafety issues. If the purified allergens are released to the market, they must be regulated by the local health norms and distributed to certified laboratories alone. Likewise, these heterologous proteins must be handled in the same way as the naturally occurring proteins would be.

In order to protect the public safety in the surroundings of the laboratory, the entrance of the working area is regulated by a digital fingertip recognition system. Also, a special lab permission is needed in order to work with any pathogenic organism. By preventing public contact with the strains we’re working with, we are trying to reduce the possibility of the release of any dangerous genetically modified organism.

Three days a week, the laboratory is used for academic purposes, which means that a considerable amount of students often visit our working areas. So, to ensure their safety, we disinfect any surface that could have risk of being contaminated. Regarding the use of dangerous compounds, i.e. ethidium bromide and acrylamide, we work with this substances in a different laboratory in order to avoid leaving any remnants that could harm the students.

o Environmental Safety

In the given case that one of our genetically modified organisms was released unintentionally to the environment, we believe it would present no significant threat, as the laboratory strains are not bound to survive for a long period of time outside regulated environments.

The RiAFP produced by our E. coli strain is expressed in low quantities, and in the worst case scenario, it could help the bacteria survive in colder places; but this is highly unlikely to happen, as E.coli has limited capacity of growth at low temperatures since all its enzymes are mesophilic. In the case of P.pastoris, the probability of survival is even lower, as this organism is more sensitive to environment conditions.

Also, some of the experimentation processes include using toxic substances, and most all of them are related to E. coli or P. pastoris culture. For this reason, we needed a standardized protocol of waste disposal. When the wastes were originated from a biological or pathological source, we placed them in a biohazard waste bag, which would after be sterilized at the autoclave and disposed in a safe place. EtBr and acrylamide wastes were treated separately from each other and from biohazard wastes, this because of the different properties they have.

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

We will, eventually, as the parts we made this year are allergens; and they can be harmful to some people, specially those who have presented an allergenic previous response to maize, dust mite and/or honey bees’ sting. Even if someone has not presented an allergenic response, it is important to know that this kind of response can be developed even with the non-recombinant version of the proteins.

o How did you manage to handle the safety issue?

Before we started working on the allergen project, we made sure that none of the people that would be directly operating the cells cultures and the samples presented any kind of allergic response. Besides, we followed the established norms and procedures for our laboratories.

As an extra precautionary measure, any member of our team handling allergen samples or modified P. pastoris cultures, had the obligation of wearing a procedure mask at all times.

o How could other teams learn from your experience?

Before any other team attempts to works with any of our Biobricks; we highly encourage them to pass through a medical examination to verify that none of the members suffers from aggressive immune response.

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

o If yes, what does your local biosafety group think about your project?

o If no, which specific biosafety rules or guidelines do you have to consider in your country?

Due to Human Resources issues, our institution does not posses an officially certified committee for the moment. Nevertheless, a new biosafety group is being established and is now in the way to certification.

There do exist some laws in Mexico that regulate the use and manipulation of GMOs, nevertheless they focus on genetically modified plants. Most of the safety issues and precautions listed by said laws, take in account how to care the genetically modified crops and how they should be tested (Cámara de diputados, 2012). The CIBIOGEM (Comisión Intersecretarial de Bioseguridad de los Organismos Genéticamente Modificados) was recently founded for the regulation in matters of Biotechnology, some of its functions, besides dealing with the legal side of the biotechnological activity, include sharing biotech information among people and the regulation of biosafety for kids.

2. 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?

Some ideas future iGEM competitors could implement are:

1. A linearized plasmid with not only an antibiotic resistance, but also an anti-repressor dependant-gene. This way, the plasmid would have a selectivity marker with the antibiotic resistance but will also include a repressible repressor protein (that could repress a vital characteristic constitutively expressed). This will make the host dependant to a substance that represses this gene, thus making it more difficult for the GMO to survive outside the laboratory.

2. An inductor-dependant strain that would not be able to survive “out of the lab” conditions.

We strongly believe the 2nd option (inductor-dependant strain) would imply safer biosafety conditions for every lab, not only iGEM competitors. Variations or sub-strains could derivate from this one making them (DE3), amber suppresors or any other variation.

References:

LEY de Bioseguridad de Organismos Genéticamente Modificados. (18 de March de 2005). Obtenido de http://www.salud.gob.mx/unidades/cdi/nom/compi/ley180305.html

CÁMARA DE DIPUTADOS DEL H. CONGRESO DE LA UNIÓN. (26 de April de 2012). REGLAMENTO DE LA LEY GENERAL DEL EQUILIBRIO ECOLÓGICO Y LA PROTECCIÓN. Obtenido de http://www.cibiogem.gob.mx/Norm_leyes/Documents/normatividad-SEMARNAT/REGLAM_LGEEPA_IA.pdf}

Rafael Camacho Carranza, C. E. (s.f.). Manual de Procedimientos de Bioseguridad. Obtenido de Comisión de Bioseguridad Instituto de Investigaciones Biomédicas y Universidad Nacional Autónoma de México: http://www.biomedicas.unam.mx/_administracion/_unidades_apoyo_inst/manual_bioseguridad.pdf

V Balamurugan, G. R. (2007). Pichia pastoris: A notable heterologous expression system for the production of . Indian Journal of Biotechnology , 175-186.

We are working in two different projects, thus, in order to assess the risk factor, we consider appropriate to analyze their individual issues in terms of biosafety. First of all, it is important to cover a brief description of both the materials used in the projects, and the organisms employed and their characteristics.

It is important to understand that we are always exposed to risk and no experiment is 100% risk free. Nevertheless, our job is to minimize the probability of being exposed to any danger.

During the realization of the projects we did the following procedures:

  • Miniprep (plasmidic DNA extraction)
  • Agarose gel DNA electroforesis
  • E.coli Transformation by CaCl2
  • P.pastoris Transformation by electroporation and LiAc
  • Inducible promoter induction with L-Arabinose, IPTG and MetOH
  • SDS-PAGE
  • Cell lysis and Protein Extraction
  • CFU count by dilution

The realization of the named protocols implies the use of some hazardous materials. These substances require special treatment and disposal. In our protocols we use the following dangerous substances:

  • Acrylamide
  • Ethidium Bromide
  • LiAc
  • DTT
  • Acetic acid
  • Chlorine
  • HCl
  • NaAC
  • NaOH
  • β-Mercaptoethanol
  • SDS
  • Coomassie Blue

For us to use this kind of substances some security protocols are needed to be followed.

  1. Miniprep (plasmidic DNA extraction) uses solutions with EDTA, these solutions are 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. Also solutions using NaOH and NaAc are made under a laminar flow hood to prevent any kind of dangerous inhalation.
  2. During the Agarose gel electrophoresis the dangerous part come when the researcher has to use Ethidium Bromide to reveal the gel. Ethidium Bromide intercalates into the DNA double strand, as a strong intercalator is mutagenic and carcinogen. To work with Ethidium Bromide, it is needed to designate a special workplace; also you need to restrict the number of people to the minimum. To keep the biosafety we followed all of the recommendations, also we used nitrile gloves that are less permeable to EtBr than latex gloves. Finally, we designated a special disposal bin for all EtBr–related residues.
  3. In the E.coli Transformation by CaCl2 protocol, we are exposed to the E.coli non-transformed strains. To prevent any escape to the environment of these strains, 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 15psi for 15 minutes.
  4. P.pastoris Transformation by electroporation and LiAc protocol uses several different dangerous substances, namely DTT and LiAc. Both substances are known for having effects on the central nervous system, so for our safety, we prepare small aliquots enough for the rest of the project. For additional protection, its use is restricted to a biosecurity chamber and its manipulation is only permitted when wearing nitrile gloves. Furthermore, this protocol generates biological wastes (P.pastoris) which are disposed as previously detailed.
  5. Induction protocols consist in adding a specific substance to a cell culture for it to start the production of a specific protein. In our case, our inductors are L-Arabinose, IPTG and Methanol. Inductors have no hazardous properties besides the flammable characteristics of methanol. To prevent any damage by inhalation or any possible fire, the preparation of methanol is done in a gas extraction cabinet. Also, its sterilization is done by filtration to prevent any fire or explosion.
  6. SDS-PAGE is very usefull tool at the time when we need to make a protein analysis by size, nevertheless it is full of hazardous materials. To begin with the SDS-PAGE, you need to prepare the polyacrylamide gel. This is done by adding TEMED and PSA to Acrylamide. TEMED and PSA 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. It is important to assign a separate special workspace for any procedure using these substances.

To prepare the protein sample, B-mercaptoethanol and SDS are needed, and both have toxic effects, especially B-mercaptoethanol. To keep our biosafety standards, we minimize the time of exposure to any of this materials, also we work using always nitrile gloves, and in our designated space.

After the preparation of the SDS-PAGE and the running of the gel, you need to stain with Coomassie Blue. As some of the acrylamide may have not polymerized yet, we keep our gloves on, especially because of the Coomassie staining. Coomassie Blue is a known flammable toxin that targets some of our organs.

For the Coomassie Blue to stain with more definition it is recommended to use a “fixating solution”, which contains methanol, acetic acid and water. For the preparation of this solution, an air-extraction cabinet is needed. Finally, we dispose of all the generated wastes in a bin we labeled “PAGE wastes”, separated from our normal wastes.

The CFU counting technique has no hazardous effects for the researcher. Nevertheless, it is important to minimize the probability of the transformed E.coli strains escaping the research area. For that purpose, all the inoculations are done in a biosecurity chamber and wearing latex or nitrile gloves. The counting is done in a special workplace using a Quebec colony counter. When the counting is done, every waste is disposed in a biohazard bag and autoclaved as described before.

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