Team:Tec-Monterrey/antifreeze/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.

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.

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.

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.

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.

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.

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.

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.

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.