Difference between revisions of "Team:Grenoble-Alpes/safety form"

Organisms used

Pseudomonas aeruginosa is an opportunist pathogen against humans and is the most commonly used strain in the laboratory. It is a gram-negative bacterium with a DNA of about 6.3 Kbp length.

We are working on attenuated bacterial strains such as Pseudomonas aeruginosa PAO1. Indeed, their virulence genes have been removed to make them non-pathogenic for humans.

For our proof of concept, we are using a synthetic fragment of the bacterium's DNA. So here, the bacteria itself is not used here.

However, we use those bacterias to show the efficiency of the bacteriophages as they need to be as specific as possible to a bacterium. So, we manipulate bacteriophages which are viruses that only infect bacteria in the most specific way. The bacteriophages we are using are PAKP3 and HER18. PAKP3 is a specific bacteriophage against Pseudomonas aeruginosa PAK strain. HER18 is specific to Pseudomonas aeruginosa PAO1.

All our manipulations regarding biobricks will be done using the strain Escherichia coli DH5alpha and TOP10. Indeed, we are transforming them so that fluorescence is emitted and detected.

Risks to the community or the environment if they escape the lab

Regarding bacteriophage as it is the most abundant organism on earth, we can find it everywhere. Therefore, if it escapes the lab it will not have a huge impact on ourselves, on the community or on the environment. Bacteriophages don't attack anything else than bacteria so the biggest impact is if we contaminate the laboratory.

There are also risks when using Pseudomonas aeruginosa as they are pathogenic for humans. This is why we will use attenuated bacterial strains. As with bacteriophages they can also contaminate the lab.

Moreover, we are using DH5alpha or TOP10 but they are not pathogenic for humans.

Finally, by not managing our biological waste it could be spread out in the environment which could stay in the soil or in groundwater.

Chassis species used in the project

All the bacterial transformations are carried out using Escherichia coli DH5aplha or TOP 10 (Escherichia coli). And our final detection device will use Top 10.

Risks caused by the chassis to the community or the environment if they escape the lab

We use DH5alpha and TOP10 which are engineered E. coli cells to maximize transformation efficiency. Moreover, these Escherichia coli strains do not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. They are considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

So the risks are minimal when it escapes the laboratory. Furthermore, they need to be stored at -80°C to stay efficient for transformation so staying at room temperature it will affect their efficiency and even make them unusable.

Experiments carried out with our organisms and parts

Our plasmid DNA probe is bio-informatically designed. The fraction of the plasmid DNA probe which recognizes our target will be ordered from a supplier. In addition, we will also design the Pseudomonas aeruginosa PAO1 specific gene fragments and order it online using IDT. For our proof of concept, we will use attenuated strains of Pseudomonas aeruginosa and isolate fragments of interest.

We will build a probe (plasmid) that detects a DNA fragment of Pseudomonas aeruginosa. After hybridization, TOP10 bacteria will be transformed with the plasmid probe. If the hybridization has occurred, TOP10 bacterias will be fluorescent.

We will use bacteriophages: HER18, PAK-P3 and T5 that are capable of lysing the bacteria it is specific to. We first amplify then, purify and finally concentrate them. The two last experiments are done together (in one experiment). Titration of the bacteriophages is also done to evaluate the concentration we got. PAK-P3 and T5 are only used to show their specificity to a bacteria.

To do the experiments on bacteriophages we need their specific bacteria so we manipulate: P. aeruginosa PAO1 and Escherichia coli F. We will finally extract the DNA of the bacteria PAO1 released after bacteriophages lyse it.

Potential risks caused by the experiments

No real risks could arise from our experiments as the main experiments are bacterial transformations, PCR, enzymatic digestion and genetic engineering. The only risk could be bacterial contaminations of the environment and so spreading of these bacteria and their DNA to the community and to other bacteria.

Regarding the bacteriophages experiments, no big risks are related to it as we can find them everywhere. The only risks might be if you manipulate a bacteriophage and contaminate a lab which works on the related bacterium.

The last risks that may arise from our experiments concern manipulations of Pseudomonas aeruginosa PAO1 : a Risk Group 2 organism. That means they can cause diseases in humans but are unlikely to spread to the environment, they are safe for the community, and prophylaxis or effective treatments against them are known.

Potential places where the project could be used

• In a factory

• In a small enclosed device

• Other (in a hospital setting)

Potential safety, security or ethical risks involved with such a use

Our device will contain DNA, bacteria and bacteriophages. To ensure full protection to users the components of our device such as DNA, bacteria and bacteriophages will be lyophilized or dried in the device.

Moreover, we must enlighten the public on bacteriophages as a therapy for the future. However, bacteriophages are viruses, by this, it might be difficult for people to consider them to be harmless.

Finally, in our project, there is no ethical risk.