Team:Grenoble-Alpes/safety form

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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 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.


Experts that helped to manage the identified risks

First we had a training on health and safety with a specialised scientist. In our labs, Sophie the laboratory technician is there to help us with reactives, some experiments and materials demand. Moreover, Sophie introduced us the laboratory, explained how machines work. In addition, our supervisors, Mr. Pierre Cavailles and Audrey Le Gouellec are teacher-researcher who accompanied us and gave us advices. These people are essential because of their experiences and expertise, they allow us to be in good working conditions and safe. Our supervisors are microbiologists, so they are familiar with the experimental procedures we use. In addition, they have been working for at least 5 years in the laboratory, so they have the reflexes and habits of work to optimize handling and safety.

Rules that helped to manage the identified risks

The rules to be followed for the manipulations inside a laboratory are given by the Cofrac.

Topics that we learned about in our safety training.

  • Lab access and rules (including appropriate clothing, eating and drinking, etc.)

  • Responsible individuals (such as lab or departmental specialist or institutional biosafety officer)

  • Differences between biosafety levels

  • Biosafety equipment (such as biosafety cabinets)

  • Good microbial technique (such as lab practices)

  • Disinfection and sterilization

  • Emergency procedures

  • Transport rules

  • Physical biosecurity

  • Personnel biosecurity

  • Chemicals, fire and electrical safety

Work areas we use to handle biological materials

  • Open bench

  • Biosafety cabinet (please note there are important differences between biosafety cabinets and laminar flow hoods / clean benches. iGEM encourages the use of biosafety cabinets but discourages the use of laminar flow hoods or clean benches)

Biosafety level of our lab

We have several different lab spaces with different biosafety Levels. We have both a level 1 and level 2 spaces. We have a level 1 laboratory bench and biological safety cabinet and a level 2 biological safety cabinet in another lab.

How rules, training, containment and other procedures and practices helped to manage the identified risks

The first risks that can be managed by using the training and practices we had are the waste management to reduce the impact of its release in the environment.

Moreover, what we have done to manage the bacteria transformation with a resistance cassette is that we worked under sterilized condition using gloves, blouse and using a microbiological cabinet.

We are also using bacterial strains that are attenuated to make them less susceptible to human infections.

All manipulations concerning the pathogenic strain Pseudomonas aeruginosa PAO1 were carried out in level 2 laboratory.
A risk prevention plan has been done before any experiment. It has been dictated the instruction for safety and management of infectious waste. These are rejected in "DASRI" (infectious clinical waste) bins.
In addition, a professional was present throughout the experiments to ensure compliance with the safety instructions in the lab. These include the wearing of glasses, gloves, blouse and overshoes from the entrance in the lab (passage in an airlock); and the compulsory wearing of safety cuff for handling under level 2 Biological Safety Cabinet.