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Team:Colombia/Safety
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1. Would the materials used in your project and/or your final product pose:
Risks to the safety and health of team members or others in the lab?
Working in the lab always has risks for people but we will take action to minimize any risk, some of the precautions we take are listed here: • The microorganisms will be grown in petri dishes, sealed completely during bacterial growth, thus preventing their escape into stages of experimentation. • Inoculation experiments will be 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 cabinet will be used for minimize the possibility of spread of microorganisms. • All students involved with the project will be informed of the established security arrangement and sign a commitment to strictly comply the rules. • Also, prior to the beginning of the experimental procedures, all of the team members were given a course of biosafety, focused mainly on biological hazards, chemical reagents and general BSL-1 laboratory safety concerns and security procedures.
The system design tests will be conducted within a laboratory of biosafety level 1, and we will 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 will be making are routinely in most academic laboratories in the world. For more information visit Escherichia coli
Known toxic chemical reagents and hazardous physical agents:
Ethidium bromide
An intercalating agent, toxic and suspected mutagenic, is used widely used as a nucleic acid fluorescent stain in molecular biology, in our project Ethidium bromide will be used only in electrophoretic procedures. Some of the precautions we take when working with this mutagenic agent are always wearing nitrile gloves and working in a delimitated area, avoiding direct skin contact an inhalation, specially isolated to prevent involuntary exposure of laboratory coworkers with Ethidium bromide.
Ultraviolet light
Is an electromagnetic radiation with a wavelenght shorter that of visible light. We will use UV light in the biological safety and laminar flow cabinets for sterilization and decontamination purposes; also, UV-light would be used for visualization of the stained DNA in the electrophoretic gels.
In order to prevent accidental exposition to UV radiation, precautions would be taken, and when necessary special safety lenses would be used, also we will 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 of the University for Guidelines for Integral Solid Waste Management of the Medical and Occupational Health Department at the Universidad de los Andes.
Risks to the safety and health of the general public if released by design or accident?
As previously mentioned, K-12 strains are not pathogenic neither for humans, animals or 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. 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 (Balali-Mood, 1981); as long a salicylic acid and its derivate methyl salicylate production by the bacteria plays a key role in our circuit, we evaluate the possible risk and take some considerations into account:
1. 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.
2. Salicylic acid production is under tightly control of a negative feed-back.
3. Even when ingestion of methyl salicylate is more toxic than salycilic acid (because it is more rapidly absorbed) bacteria production won’t overcome a concentration of 1,95ug/ml as long as greatest concentrations have bactericidal effects, 400 times less than required to cause toxicity.
4. 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 infinitely 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.
Risks to environmental quality if released by design or accident?
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 country field.
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 in 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 blastn 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 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 in color markers and even more important 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.
Risks to security through malicious use by individuals, groups or states?
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 fungi infection and that these modifications do not have any danger to people, plants, animals, or environment, even when we work with some pathogenicity processes related proteins, and produce Salicylic acid which may be toxic in high concentrations, our circuit is tightly control by positive and negative feedbacks, and none of these proteins are constitutively expressed, only responds to chitin presence then our project does not have any risk to security through malicious use by individuals, groups or states.
3.Under what biosafety provisions will / do you operate?
a. Does your institution have its own biosafety rules and if so what are they?
Specifically, are any parts or devices in your project associated with (or known to cause):
Pathogenicity, infectivity, or toxicity?
Despite chiA and chiP genes are involved in the sense and metabolism of chitin they also may be related to pathogenicity processes in different Vibrio strains, neither of both 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)
Threats to environmental quality?
No
Security concerns?
No
