Explanation

Before explaining what was done, here are defined some terms.

A risk

It is a possibility that a dangerous phenomenon can cause damage to a given target.

A risk assessment

A risk assessment tries to define all the present risks, evaluates their probability and the severity of each one.

Safety and Security

A difference should be made between Safety and Security. Safety intends to reduce risks in terms of accidents, incidents, damage due to them and consequences of these activities on health. On the other hand, Security deals with the misuse of devices and voluntary acts which create risks for others. We only worked on the safety during the project.

The context

We worked in the CIME-Nanotech. The laboratory belongs to the INPG safety department which took care of the team during the project. This department has the following structure:

ACMO: “Agent Chargé de la Mise en Oeuvre des règles d’hygiène et de sécurité “(Officer Responsible for Implementation of hygiene and safety)

We contacted the safety officer and explained to her what we indented to do. At first sight she was a bit frightened by the work, and she wanted to know exactly how we managed to reduce risks. Therefore one student of the group dealt with the safety assessment.

To prevent any problems with the team, each member signed a regulation that compelled us to respect some rules:

• Work between 7 A.M. and 7 P.M. because of safety problems
• Use and do not lend the personal badge which permits to enter in the laboratory
• Do not work alone in the laboratory
• Do not eat nor drink in the laboratory
• Do not smoke in the laboratory
• Use good laboratory practices
• All products must be tagged and a stock status has to be regularly updated

To reach those obligations we have done a safety inspection with the help of the Safety department. The goal was to inform people about risks in presence and rules to respect. Moreover we wrote a safety manual of good laboratory practices available here.

What has been done

In the context of iGEM we have:

• Spotted the main risks
• Reduced the chemical risk
• Checked if the UV risk was under control
• Improved the safety in biology

Spotting main risks

In order to improve the safety of our project and to be efficient, we focused first on spotting the main risks due to laboratory manipulations.

To this end, we made an inventory of all devices and manipulations which would be used and done.



Chemical risk




Explosion risk




Mechanical risk



Risk due to devices under pressure



Risk related to ionizing radiation




Biological risk





We made a parallel between our risk assessment and the one made by the safety department of the CIME.

How to read a risk assessment grid?

Here you have an example. What we were looking at, is the value in the column grade associated to each risk. The higher it is the more predominant is the risk.

This study led us to three main risks:

• The chemical risk
• The risk related to ionizing radiation (UV)
• And the biological risk

Reduction of the chemical risk

It appears that it is essentially the revelation of the DNA with the ethidium bromide (EtBr) which is dangerous. Indeed, EtBr is a mutagen product of level 2, meaning that its mutagenicity effect was confirmed on animals at low concentration (no exposure limit value is given by the manufacturer). We must therefore follow the precautionary principle, especially because it is stored in its purest form.

Regarding contamination, direct contact with skin is the only way to be exposed. Indeed, the saturation vapor pressure of ethidium bromide is relatively low. In theory no exposure occurs.

How are chemical products classified

In the laboratory the risk is taken very seriously. An area is exclusively devoted for its use. Moreover nitrile gloves are available as well as full face protections. Finally a specific dustbin is in place for disposal. (For more details see the safety manual practice).

To find information, we sought the EtBr MSDS. We first wanted to know where EtBr was, to check if there is any risk of being in contact with the product. To detect its presence we used an UltraViolet (UV) lamp. Red circled areas were positively controlled.



To prevent the use of EtBr, we tried to find substitutes. We found the NANCY-520 and the SYBER-SAFE. However the NANCY 520 could not be used because we did not have the appropriate wavelength on our lamp. As far as the SYBER-SAFE® is concerned, it appears to be safer, but the revelation takes too much time. It did not meet our needs. Therefore we did not change the product.

Then we used regulation and we managed to improve the workstation via three ways:

• Work organization
• Technic
• Training

Our work led us to put in place these things:



Moreover a protocol was set up. Here you have a video, that shows the main things to know. (in a fun way)

Check if the UV risk is under control

The revelation needs to use an UV lamp (312nm at 48W). But biologists need to be very close to the lamp to cut gels. Therefore we checked if PPE (Personal Protective Equipment) were efficient. The verification confirmed that our equipment meets the standard requirements, thus we can say that the risk is under control .

Improve the safety in biology

Synthetic biology is a new field in biology and intends to develop new organisms with new genetic systems. Therefore it could also be a new way of developing technologies. However for every new activity, there are new risks.

The dangerous effect assessment needs to be split into two parts. The first one deals with direct effect due to the recognized pathogenicity of the biological elements that we used. The second part takes care of potential effects that could be due to genetic modifications.



The study of the direct effect wasn’t necessary as we were working with non-pathogenic organisms. Indeed, we used Escherichia coli referenced as BW25113 (for more information see the link here ). Moreover, all biological parts are coming from non-pathogenic organisms or are not coding any dangerous elements. Our system does not seem to be dangerous, as it produces no toxins and even the quorum sensing (cAMP) does not present any proven risk. Nonetheless, the precautionary principles oblige us to decontaminate the biological waste . Besides, we tried to reduce the spreading of microorganisms by using good laboratory practices As far as the second part is concerned, there is a real difficulty to know what could happen.

But we tried to work on the issue via two different approaches.

First approach

The first thing on which we focused was the spreading of microorganisms. Indeed, we are not able to know if potential effects can occur, thus we tried to figure out how we could get in contact with microorganisms.

To achieve this characterization, we followed those steps



Thanks to their study we focused on the ones where microorganisms are present. By using a grid of assessment we found steps that generate possible aerosols. This grid is available here.

This approach led us to do a failure modes and effects analysis.

what is a failure modes and effect analysis

It is a technique which consists in focusing on each equipment and material used. The goal is to understand what happens when something goes wrong. The result is given as following.
Finally we put all important information in a fault tree analysis.

What is a fault tree analysis

A fault tree analysis is another technique, which consists in listing all elements that can cause a dreaded phenomenon. These events are then linked thanks to Boolean logic connectors. The assembly is then placed in a scheme which allows you to draw all the scenarios involved in the dreaded phenomenon or situation.

In the following fault tree analysis we represented the steps in which we could have a potential propagation of microorganisms.



The tree emphasizes the fact that we have three different kinds of situations which could lead to microorganisms propagation:

• normal situations
• situations caused by human errors, or failures to internal manipulations
• situations induced by events unrelated to manipulations’ mistakes

Note that according to this tree, the spread of bacteria in the laboratory is caused by manipulation under flow hoods. Meaning that in theory, people present during handling are exposed to bacteria. Nevertheless, this study does not take into account the concentration at which individuals are exposed, and how it is distributed in the laboratory. It only gives indications about the possible causes of any release.

Second approach

To deal with the issue of biological risks, we focused our work on the first step of prevention which is the identification of all information to assess the risk.
Prevention can only be put in place thanks to feedbacks. Thus, we needed a device or an organization which enabled us to record them.

In the context of iGEM, we had an idea that we called the BioBrick Safety Sheet (BSS). This sheet aims at:

• Improving the way of collecting information on how BioBricks can be used safely
• Assisting biologists in their work
• Establishing a standardized system to collect information in synthetic biology

To develop this sheet, we established a list of information that it should contain. Regarding biology and modelling; it should contain, basic information about the constituents of BioBricks and their characterization. Concerning the prevention when using the BioBrick, it should identify environments in which it has already been used and problems encountered. Finally, the sheet should be updated after each use of the BioBrick.

Here is a link towards the template of the BSS.

In our human practice project we asked other teams to give us feedbacks on our BSS in order to improve it.