Biosafety Provisions

Institutional Regulations

Our project safety procedures are governed by the general regulations of the University regarding the proper standard rules and safety guidelines for laboratory practices.

Teaching and research laboratories in the university are specially regulated accordingly to the dependence they belong to. In our case, the Faculty of Sciences Department of Biological Sciences and Department of Physics.

The specific guidelines for the laboratory work at the Department of Biological Sciences are available online (in Spanish only) [http://micro.uniandes.edu.co/MICUA_/Docencia_files/Reglamento%20DCB%202010.pdf Biosafety Guidelines].

Our main workspaces are the [http://lamfu.uniandes.edu.co/HOME/Home.html LAMFU] and the [http://biofisica.uniandes.edu.co/ Biophysics] facilities, BSL2 research laboratories, but also we occasionally work on several BSL1 teaching laboratories (Department of Biological Sciences).

Institutional Biosafety Committees

There is the Parietal Committee for Occupational Health (COPASO), regulated by the Colombian Constitution (Resolution Number 2013, Year 1989). The information regarding the functions and regulations of the COPASO committee is available [http://tramites.minproteccionsocial.gov.co/tramitesservicios/copaso/copaso.aspx online] (Spanish only).

There is also the [http://investigaciones.uniandes.edu.co/index.php?option=com_content&task=blogcategory&id=56&Itemid=65 Bioethical Committee] of the Faculty of Sciences, to whom we have presented our project proposal. Although we are still waiting for a response from the committee, our research methods and safety precautions are well within the regulations of the Committee.

Biosafety Training & Work Precautions

All team members were required to attend safety training focused on:

• General biosafety principles.

• The proper use of containment equipment (Biological safety (laminar flow) cabinets and delimited working space when required) as well as personal protective equipment (PPE) (White coat and gloves).

• Risk assessment of experiments involving: Recombinant DNA, ethidium bromide, other biohazardous materials.

• Proper Biological waste disposal.

Our main concern was to work with the appropriate safety guidelines for working in both research and teaching laboratories (BL1-BL2) in order to minimize exposure to hazardous agents. We also work with the participating laboratories´ procedures to minimize waste and encourage use of reusable glassware instead of disposable plastic.

Safety Considerations: Hazardous reagents and Biohazards

Health risks & Personal safety

Working in the lab always has implicit risks, but we have taken action to minimize them. Some of the precautions we take are listed here:

• The microorganisms are grown in large petri dishes that are sealed completely during bacterial growth, thus preventing their escape.

• Inoculation experiments are conducted on plants that are isolated in a greenhouse. Plants will be kept for a minimum time of experimentation and will be discarded as biohazard material immediately after the experiments.

• Equipment such as biological safety cabinet class I and laminar flow cabinets are used regularly to minimize the possibility of spread of microorganisms by recently trained students.

• All students involved with the project were informed of the established security arrangement and signed a commitment to strictly comply with the rules.

• Also, prior to the beginning of the experimental procedures, all of the team members were given a course on biosafety, focused mainly on biological hazards, chemical reagents and general BSL-1 laboratory safety concerns and security procedures.

The system design tests are conducted within a laboratory of biosafety level 1, and we use the following materials and microorganisms strains:

Bacterial strains:

Escherichia coli K12

According to the Final Risk Assessment of the U.S. Environmental Protection Agency (EPA) (1997), Escherichia coli K-12 are not likely to pose any risk to human or animal health, to plants or to other microorganisms. Any concerns in terms of health considerations are mitigated by its poor ability to disseminate, colonize the colon and establish infections in a murine model (Smith et al., 2010).

Similar results have been observed in chickens, pigs and calves. In addition the probability is low that the insertion of genetic material meeting the EPA’s criteria (introduced genetic material must be limited in size, well characterized, free of certain nucleotide sequences, and poorly mobilizable) into strains of E. coli K-12 will change their behavior so that they would acquire the potential for causing adverse effects.

The use of good laboratory practices in the research setting must be enough to minimize the potential of E. coli K-12 strains risk of colonize the human colon. The genetic manipulations that we are making are routine in most academic laboratories in the world. For more information visit [http://epa.gov/biotech_rule/pubs/fra/fra004.htm Escherichia coli].

Aliivibrio fischerii

Aliivibrio fischerii is a marine bacterium found mostly in symbiotic relationships with squids. It is widely used as a model organism for bioluminiscence and quorum sensing, and not considered dangerous to humans.

We used an Aliivibrio fischeri strain ES114 provided by Dr. Eva Ziegelhoffer from the Microbial Sciences Building of the University of Wisconsin - Madison (1550 Linden Drive, Madison, WI, 53706).

Ralstonia solanacearum

Ralstonia solanacearum is a very important pathogen of a wide variety of plants, is found in many different soil types and contaminate waterbodies. Despite it is not consider a risk for human health it is consider very dangerous for the ecosystem. Handling of this organism was performed exclusively by qualified and experienced personal in flow chamber where waste and residues were carefully handled. Special care was taken in not releasing it into the ecosystem through the drainage system. We used DNA from Ralstonia solanacearum instead of the living organism to reduce the chance of releasing the pathogen, also we used a mutant for phcS, the 3OH-PAME synthetase, it have been shown that an impairing of the Quorum Sensing ability reduces drastically the fitness and pathogenicity of the bacterium().

Known toxic chemical reagents and hazardous physical agents:

Ethidium bromide

Since ethidium bromide is a very toxic agent, we have changed our nucleic acid staining molecules to [http://www.biotium.com/product/product_info/Protocol/PI-41001.pdf Biotium GelRed] and [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/DNA-Stains/SYBR-Safe.htmlInvitrogen SYBR Safe]. Both of these claim to be significantly less toxic and better for the environment. Actually, both may have direct disposal into the wastewater drainage systems.

Ultraviolet light

It is a type of electromagnetic radiation with a shorter wavelength shorter than that of visible light. We use UV in the biological safety and laminar flow cabinets for sterilization and decontamination purposes. Also, UV radiation is used for visualization of the stained DNA in the electrophoretic gels.

In order to prevent accidental exposition to UV radiation, precautions are taken and when necessary, special safety lenses are used. We always use UV-blocking shields when visualizing electrophoretic gels in UV light.

All other toxic and chemicals will be handled to avoid direct contact, and observing the proper safety procedures, additionally, all chemicals reagents and biological materials will be disposed only in the designated biohazard receptacles, following the regulations for [http://departamentomedico.uniandes.edu.co//Documentos/Salud_Ocupacional/Procedimientomanejoresiduossolidos.pdf Guidelines for Integral Solid Waste Management] of the Medical and Occupational Health Department at the Universidad de los Andes.

Public Health

As previously mentioned, K-12 strains are not pathogenic for humans, animals nor plants, and the genetic modifications made in the laboratory won’t improve in this particular case the ability of the bacteria to prejudice the human health, so the risks in an accident are minimal. It is also important to mention that no human nor animal pathogenic strains of bacteria are going to be used in the current research; nevertheless the national directions regarding Genetically Engineered Organisms (GMOs) will be followed.

Besides that, exposure to high levels of salicylic acid may cause irritation or burn healthy skin, and ingestion of concentration above 800ug/mL is severely toxic and may be lethal (Proudfoot, 1983); since salicylic acid and its derivate methyl salicylate´s production by the bacteria play a key role in our circuit, we evaluate the possible risk and take some considerations into account:

• Salicylic acid and methyl salicylate only have adverse effects when eaten, inhalation may cause irritation of mucous membrane and upper respiratory track but never lead to death.

• Salicylic acid production is under tight control by a negative feedback.

• Even though ingestion of methyl salicylate is more toxic than salycilic acid (because it is more rapidly absorbed) bacteria production won’t surpass a concentration of 1,95ug/ml because greater concentrations have bactericidal effects, and this is 400 times less than required to cause toxicity.

• In case of a breakdown of the regulation control system of the circuit, overproduction of salicylic acid and its derivate methyl salicylate won’t enhance indefinitely the production of salicylic acid in the plant. Plants have a strict control of their defense systems and hormones production, as excess of them may have side effects on growth and defense functions. Therefore, leaves and fruit will never reach concentration values above 800ug/mL.

It is important to mention that, although our project proposes an agricultural application of the strains generated, in this stage of the investigation we will not do toxicity tests in animals or humans of the genetically modified organism.

Environmental Quality

Although the initial stages of our research are mainly laboratory-based, the goal is to implement our system directly in coffee plantations. Because of this the acquisition of chitin degradation related genes and the kanamycin and ampicillin resistance cassettes extend its host range and increase its fitness, there may be a low concern about releasing our GMO K-12 in the fields during testing.

In order to minimize the side effects of introducing beneficial traits for the adaptation to different environments in the bacteria, we take into account the EPA’s criteria for genetic material,

• We design primers and amplify known sequences from Vibrio fischeri strain ES114, which is naturally found in a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes.

• These sequences have been previously characterized and used for research.

• Even when some of these proteins can be related to pathogenicity processes, none of them alone can cause disease, as they are not toxins.

• To avoid cloning unknown or hidden pathogenicity related proteins we amplify the majority of genes from the start codon to the stop codon, and a Blast of the sequences was carried out to confirm results and discard any pathogenicity trait.

• Cloning was performed in vectors without a mobilizable origin or Tra region avoiding horizontal transfer of those genes in the environment.

In addition we made a growth curve in planta using coffee plants, and the result confirm that our modified K-12 poorly colonize and disseminate in coffee leaves, so in case of accident our GMO K-12 would barely spread in the environment.

We are also planning to develop a strategy to monitory the presence of the modified bacteria in the crops fields based on color markers and even more importantly we hope to build our bacterial-based detection system on organisms representing low-to-no risk for the normal microbiota of the plantations and innocuous for human consumption in the future.

Security Issues

Taking into account that the genetic modifications we will make to the Escherichia coli K-12 strain are focused to improve the ability of plants to survive a fungal infection and that these modifications do not pose any danger to people, plants, animals, or the environment; and even when we work with some pathogenicity process related proteins, and produce Salicylic acid which may be toxic in high concentrations, our circuit is tightly controlled by positive and negative feedbacks, and none of these proteins are constitutively expressed but only respond to the presence of chitin, we consider theat our project does not have any risk to security through malicious use by individuals, groups or states.

Biological Parts and Devices

chiA and chiP genes are involved in the sensing and metabolism of chitin, but in principle they also may be related to pathogenicity processes in different Vibrio strains. However, neither of them can cause disease by themselves, as they are not toxins.

ChiA is an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-enconded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin (Meibom et al., 2003).

ChiP is a chitoporine found in Vibrio furnissii similar to a porin from Vibrio cholera, the Salmonella typhimurium phoE porin, the E.coli phoE porin, and to a wide variety of other outer membrane proteins and porins. It appeared as if the gene immediately following the porin structural gene encodes a toxic protein (Keyhani et al., 2000), so we took special care to avoid cloning it.

All other parts we use are standard promoters, RBSs, terminators and fluorescent reporters, or have no known risks.

References

1. Proudfoot, A. T. (1983). Toxicity of salicylates. The American journal of medicine, 75(5A), 99–103. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6650533

1. Smith, S. N., E. C. Hagan, M. C. Lane, and H. L. T. Mobley. 2010. Dissemination and systemic colonization of uropathogenic Escherichia coli in a murine model of bacteremia. mBio 1(5):e00262-10. doi:10.1128/mBio.00262-10.