Team:UFRGS Brazil/Safety

Safety

Overview

Our bacteria (Escherichia coli K12 MG1655) will be engineered to degrade glyphosate through specific modifications in an operon involved in phosphonate assimilation. These modifications will enhance the expression of proteins associated to transportation and processing of organophosphonates compounds. The bacteria will be contained in alginate spheres, inside a device to filter water, allowing these organisms to be in contact with the water, where they'll exert their glyphosate degrading function.

There is no identified risk from the use of this E. coli strain, even if it escapes from the laboratory, because our subject organism has little or no demonstrated hazard. E. coli K12-derived strains are well known bacteria employed in genetics and molecular biology since the 1950's. They have been modified so that they are able to survive in culture only under very specific conditions, and are unable to survive at all in the human gut or the common environment.

Our laboratory is biosafety level 1, we use open benches and biosafety cabinets.

Possible risks:

As our project deal with a genetically modified organism we deal as well with its risks, such as interactions with other organisms, outcrossing and a free life outside our product. Our group made a series of modifications aiming these risks to make sure that none affects our goal.

Experiments and its risks:

Our bacteria will be located inside alginate spheres, engineered to degrade glyphosate contained in water. We will test them simulating these conditions under laboratory environment. We shall be verifying parameters such as glyphosate degrading efficiency and survival in and outside these environments.

The main risk is the escape of engineered bacteria. Nevertheless, bacteria employed are derived from E. coli K12 that is well known to not harm humans, animals or the environment. They are auxotrophic, i.e., they depend on complex substrates to survive, found mainly in formulated microbiological media. In the unlikely event of escape from the lab, these bacteria will not survive. In addition, the introduction of a kill switch makes our bacteria die in non-lactose media. While our alginate spheres contain this substrate, so the bacteria survive, the water environment it can be released doesn't, mitigating this risk.

Safety, security or ethical risks involved:

Biosafety in Brazil is regulated by Biosafety National Technical Commission (CTNBio) attending Federal Law 11.105/2005 and related legislation. According to CTNBio, E. coli K12 strains are of the lowest risk, being organisms of Risk Group I. Our laboratories are registered and authorized by CTNBio to operate with E. coli K12 strains. Any experiment outside the lab will need the previous evaluation and authorization by CTNBio after exhaustive analysis of our proposal. Main arguments of biosafety are the following: (i) bacteria will be physically enclosed in alginate beads and beads will be restricted to polymeric barriers in a filter; (ii) E. coli strains used will be armed with kill switches; and (iii) E. coli K12 strains are auxotrophs, and depend on complex substrates to survive. Bacteria will be active for a determined time lapse as far as medium is available to them.p>

One of the professors that helped the project is an expert in biosecurity, and is a former member of CTNBio, the national institution that rules biosecurity matters in Brazil. The other PI has extensive experience in microbiology. Both of them will be personally guiding and overseeing our experiments throughout the entire project.

Rules and guidance covering the work:

Brazilian Federal Law 11.105/2005 is the main regulation concerning biosafety in Brazil. The National Technical Biosafety Commission (CTNBio) was created by this law and is the entity responsible for all activities concerning genetically modified organisms (GMOs) in the country. CTNBio presents a series of Normative Resolutions that regulate such activities. Our laboratories and PIs are recognized and authorized by CTNBio to proceed with experiments with GMOs of Risk Groups I and II, according to the Biosafety Quality Certificate Nr. 060/98. This project will employ E. coli K12 strains that belong to Risk Group I and exclusively within the laboratory. Any further experiment outside the lab will have to undergo analysis by CTNBio. All students involved in the manage of the microorganisms had received a proper biosafety training. Besides, following the good laboratory practices along with proper use of personal protective equipment inside the Biotechnology Center facilities, never leaving the laboratory with lab coats or samples used in the experiments and the correct waste disposal will help to manage the aforementioned risks.

Risk management tools:

Accident reporting; personal protective equipment; access controls; medical surveillance and a waste management system.

The biosafety training received from our PIs will guarantee that all good laboratory practices will be followed. The proper use of personal protective equipment in the Biotechnology Center is respected, never taking lab coats or samples used in experiments outside the lab. As stated before, bacteria employed in our experiments belong to E. coli K12 strains which are Risk Group I organisms. All wastes that may have contact with the microorganisms are autoclaved before disposal.

IGEM’s rules and policies:

● We do not plan to/have released any organism or product derived from our project;

● We do not plan to use nor are using any animals (including insects and invertebrates);

● We are not using any parts or organisms obtained from outside the lab or regular suppliers.

The biosafety training received from our PIs will guarantee that all good laboratory practices will be followed. The proper use of personal protective equipment in the Biotechnology Center is respected, never taking lab coats or samples used in experiments outside the lab. As stated before, bacteria employed in our experiments belong to E. coli K12 strains which are Risk Group I organisms. All wastes that may have contact with the microorganisms are autoclaved before disposal.

Safety design

During the development of our project, we always considered biosafety as one of our major concerns. Designing a bacteria able to degrade a compound of interest is not enough; we need to make sure it won’t damage an already well established natural environment, which could be a consequence of bacterial leakage. We must be certain that our bacteria won’t escape the environment designed for them. Although alginate beads can be a very tight structure for bacterial imprisonment, one cannot expect that it will solve all of our problems. Nonetheless, even if these organisms escape, a second containment mechanism will be engaged to avoid any ecological damage, since these beings will be disposed in a natural environment.

Considering that, one of our goals was to use a very utilized and promising methodology: a Kill-Switch based system. Other teams in iGEM already created and characterized several of them. Our idea is to use a system that will enable the death of an organism when a certain compound is absent in its media. How’s that? In a general way, the mechanism we employed consists in the presence of IPTG: when it is present, it will bind to lac Repressor, which will promote cI transcription. When cI is present, it will bind to the operator region downstream of its CDS, inhibiting the expression of MazF toxin. If IPTG is absent, this mechanism won’t work, and MazF production will be activated, leading to cell death. This system represents another barrier that our organisms will have to cross to be able to cause any unwanted ecological damage.