Project

Overview

Very often, the risks related to the safety of a genetically modified organism (GEO), except for a possible toxicity or pathogenicity of the organism, correspond to the spread of GEO. Most laws, "guidelines" and associations related to the Biosafety agree in trying to prevent the spread of any living modified organism. They suggest a precautionary approach whose main purpose is the protection of the environment and human health. This is clearly stipulated in the Cartagena protocol on Biosafety (international Convention on Biological Diversity): "The Protocol seeks to protect biological diversity from the potential Risks Posed by living modified organisms (LMOs) resulting and from modern biotechnology". Synthetic biology fits especially into the definition of "modern biotechnology". Indeed, biological machines designed in the iGEM competition, and more generally in synthetic biology, do not exist in nature and can be randomly introduced into existing natural biomass through undesired cross interactions.

This is why the scientific community is unanimous in saying that the "biosafety mechanisms" are essential and will ensure the neutralization of the living device created outside the laboratory conditions under which it was generated. Thus, any alleged dissemination would be impossible.

These mechanisms can be of several types, the most famous examples are the suicide genes (encoding RNAse, DNase https://2012.igem.org/Team:Paris_Bettencourt) and/or the establishment of a nutrient dependency, where the selected nutrient does not exist in the external environment.

We reviewed previous iGEM projects over the last decade and found that despite the crucial aspect of biosafety in synthetic biology and iGEM, especially for projects registered in "Environment" and "Health / Medicine" track, it happens that no biosafety mechanism is designed, or even considered. This may be due to lack of time or resources on the part of teams preferring to focus on developing their basic idea by putting safety into the background. This reason is understandable given the complexity of the challenge of Biosafety.

Our solution

In this context, the 2015 iGEM Paris Saclay team has decided to focus its project entirely on Biosafety. The goal is to provide the community with an original system able to prevent or at least significantly reduce the main danger: the spread of GEO. This system will include a chassis called SafetE.coli and can be used by all future iGEM teams, which will be a great time-saver and will give an easy access to requirements in terms of safety to all iGEMERS. In addition to this system, our project also includes a brand new encapsulation device also intended to prevent the escape of Genetically Engineered Coli.

Environment Contamination Evaluation

We decided to experimentally simulate a GEO spreading in the environment in order to evaluate the survival rate of genetically modified E. coli strains. To achieve such purpose, we collected different natural media such as soil, sea and fresh water in which we introduced growth culture of different E. coli strains. Then, each day, samples from these contaminated environments were withdrawn and plated on a selective medium to observe the evolution of the strains’ survival.

Soil Experiment CHANGER CELA SELON LE SOUS MENU

The main goal of our project is to prevent accidental dissemination in the environment of GEO, and more specifically, of Escherichia coli, the most widely used chassis in iGEM. But before elaborating sophisticated physical and biological containment systems to prevent dissemination, we thought it would be interesting to study the E. coli survival in different natural environments.

Protocols

Environments

Our experiment aims at simulating the accidental contamination of various environments with an E. coli culture grown in LB medium. For this purpose, we chose to test different environments:

• Soil, collected around the lab (GPS coordinates: 48.703073, 2.172946)
• Freshwater, collected on the University campus (GPS coordinates: 48.702986, 2.168050)
• Sea water, which came from the Atlantic Ocean (GPS coordinates: 47.227968, -2.176851)
• Filtered sea water, which came from the Atlantic Ocean
• Filtred freshwater
• Isotonic water
• Sterile water
• LB

We chose soil, freshwater and seawater to test E. coli survival because we thought that they are most common environments that can be found in nature. As a positive control, we chose to measure the growth of E. coli in LB medium in same experimental conditions.

As control for osmotic pressure on cells, we chose to examine our strains in isotonic water. To determinate if some other microorganisms or something which can be in natural water, we chose ton filtered it or not. As comparative, we use sterile water.

Strains

We next needed to choose the E. coli strain that will be used to contaminate the chosen environments. We first thought about using a strain of Escherichia coli carrying a plasmid expressing a fluorescent protein. This strain would have allowed us to evaluate easily the number of living E. coli bacteria in our samples. However, using such strain had also several drawbacks

• First of all, we must be able to differentiate our fluorescent E. coli from other auto-fluorescent microorganisms or molecules in our samples. Even if we compare our samples to negative controls, our experiment might not be very precise.
• As no selective pressure would be applied to keep the plasmid, some bacteria may lose it during the experiment.

To address these problems, we chose to use three E. coli strains, each with a different antibiotic resistance gene integrated in its genome. These three strains are mutants from the Escherichia coli strain MG1655:

• 1320 – resistant to Chloramphenicol (Cm)
• 1693 – resistant to Spectinomycin (Spectino)
• 1696 – resistant to Tetracyclin (Tetra)

We used three different strains as inserting a resistance gene in the genome of a strain may impact the fitness of this strain in the environment. Repeating the experiment with three different strains will allow us to compare the behavior of the three strains in a given environment and identify a possible influence of the gene inserted on the survival of the strain.

Medium

To count our E. coli strains, we chose to use the Mac-Conkey medium. This medium is very selective for Gram negative enteric bacilli, so Escherichia coli. It contains bile salt and crystal violet dye to inhibit Gram positive strains, and neutral red dye which turns pink all colonies able to fermentate the lactose.

Thus, by adding the appropriate antibiotic to this medium, we thought we would be able to select our strains only, although we needed to verify this hypothesis.

Preliminary Study

Our experiment relies on the hypothesis that we will be able to detect selectively our E. coli strain when plating a sample taken from our tested environments on MacConkey plates supplemented with the appropriate antibiotic. We therefore needed to test this hypothesis and verify that the natural microorganisms found in our environment samples would grow or not on our selective plates. We also decided to grow our samples on LB medium as this medium is very rich and allows the growth of many microorganisms. We wanted to see if there was a difference with the MacConkey (MCK) medium, and if we could see the growth of the natural "inhabitants" of our samples.

We chose to analyze the soil, seawater and freshwater samples. We prepared 5 dilutions (10-2 to 10-6) of a sample of our natural environment and plated 100 µl on the following media:

• LB
• LB + Spectinomycin (Spectino : 100ng/μL)
• LB + Tetracycline (Tetra : 10ng/μL)
• LB + Chloramphenicol (Cm : 20ng/μL)
• MCK
• MCK + Spectinomycin (Spectino : 100ng/μL)
• MCK + Tetracyclin (Tetra : 10ng/μL)
• MCK + Chloramphenicol (Cm : 20ng/μL)

We next incubated the plates overnight at 37°C and observed them the following day.

For the soil experiment (Figure 1. A), we observed the growth of microorganisms on LB medium for the 10-2 dilution. These micro-organisms, however, appear to be sensitive to three antibiotic tested (chloramphenicol, spectinomycin and tetracycline). They are also unable to grow on the MacConkey medium. No growth of micro-organisms was observed when samples of freshwater (Figure 1.B) or sea water (Figure 1.C) were plated on either LB or MacConkey media. This may have been expected for the seawater medium, as micro-organisms from this environment may require high salt media for growth.

Physical containment

Considering the non-negligible risk of escape of a GEO, we wanted to add an extra layer of safety in our project. Therefore, we devised a way to physically contain E. coli and other bacteria without hindering it to perform its main function. Indeed, the system we designed is a porous glass beam in which bacteria can grow, survive and carry out their function. The nutrients of the external medium, like sugars, amino acids, etc. can penetrate inside the devise but the bacteria cannot escape from it.

Thus, the SafetE.coli chassis can be in a safer manner in contact with natural environments, which is an important aspect when the project competes in iGEM competition tracks such as Environment (lien) or Health and Medicine.

Some teams include in their project a devise, more or less sophisticated, in which the GEO or their products will perform its function. Here we can cite the 2014 project of NCTU Formosa where we can found an example of such devise: The pyramid trap (lien) containing PBAN (Pheromone Biosynthesis Activating Neuropeptide) produced by a Genetically Engineered Escherichia coli. However, other teams do not have the time or means to do as well.

Thus, the 2015 Paris Saclay team project offers to all iGEM teams a fast, efficient and inexpensive way to build an impenetrable shelter for bacteria.

The system principle

The aim of the containment is to create a physical barrier which should prevent accidental spreading of bacterial culture. The idea is to make a containment where bacteria can grow but which does not allow them to be in contact with the environment. Bacteria will be contained inside silica monoliths. To enable cell growth, we found a protocol which describes how to make cavities inside silica monoliths possible.

First, cells are encapsulated in an alginate gel. The alginic acid polysaccharide is a linear copolymer of β-D-mannuronic and α-L-guluronic acid extracted from brown algae or bacteria.

The bacterial culture is mixed with sodium alginate and the solution is dropped in a chloride calcium solution. At neutral pH, carboxylic acid functions are deprotonated so that the polymer bears a global negative charge, usually compensated by sodium ions. Addition of divalent cations such as Ca²⁺ induces cross-linking of the polymer, and therefore gel formation.

Then, silica monoliths are created around the beads obtained by a sol-gel process. The formation of silica gels at room temperature from aqueous precursors was rendered possible by the development of sol-gel chemistry (Brinker and Scherrer 1990 ). The most popular precursors are silicon alkoxides Si(OR)₄ with R being an organic group (-CH₃, -C₂H₅,...). When put in contact with water , they undergo hydrolysis, creating water-soluble species bearing Si-OH silanol groups and releasing ROH alcohol molecules. A suspension of silica is mixed with sodium silicate to make a polymerization.

Finally, the gel is dissolved by using an acid which chelates Ca²⁺ so that cells have a cavity where they can grow and which can be filled with a defined growth medium.

Protocol

The first one is performed by dropwise addition of a 1.5% (w/w) sodium alginate cells suspension in a 0.1 M CaCl₂ solution. After 10 min stirring, about 3 mm diameter beads are easily collected by filtration. The calcium alginate polymer prevents cell contact with synthesis precursors. The second step consists of silicate polymerization in the presence of commercial silica nanoparticles (Ludox HS40 from Aldrich), leading to a nanoporous monolithic structure. Monoliths are prepared at room temperature by mixing 2 volumes of 1.25 M sodium silicate with 1 volume of colloidal silica and 1 volume of succinic acid (5 wt %) into a recipient containing the alginate-cells bead. Once the sol-gel polymerization reaction is completed, the stiff monolith obtained is left in contact with 0.05% potassium citrate 3h. To provide necessary nutrients to the immobilized cells, potassium citrate solution is further replaced by LB medium according to encapsulated cell strain requirements.

Results

We obtained nice beads which are properly jellified and measure about 3 mm as expected:

The silica monolith was obtained only when we let it in contact with air:

We did not have the time to test our system.

The Temperature-Based System for E. coli: SafetE.coli

Perspectives

Modeling