Safety

The projects that are made possible by iGEM are incredible in their variety and potential. While some make their bacteria smell like bananas, others have worked on astrobiology, laying down the foundations for creating micro-organisms that can tolerate the extreme conditions of space. Others yet are inventing new ways to combat real world diseases or energy problems. What needs to be remembered is that even the banana project raises safety issues, for example the possible dissemination of genetically modified organisms in the environment. This dissemination is something that is required by the other projects mentioned above, if they were ever used in an industrial context, away from their origins in tightly controlled research environments.

This is why safety considerations and an analysis of consequences need to be taken into account, should biological material from the lab be released into the environment. In the case of our project, we analyse each individual part in terms of safety issues and try to imagine how they could interact with the environment if they should ever be accidentally or intentionally released.

KillerRed

When working with proteins like KillerRed, one can quickly raise safety issues. We have been contacted by the ATOMS-Turkiye iGEM team on the possible toxicity of KillerRed, for example. This shows why it is particularly important to study possible risks arising from use of new parts like this protein.

The ATOMS-Turkiye iGEM team believe that communication about safety issues can improve overall safety levels in synthetic biology. Their outreach consisted in accumulating information on other teams' safety issues with their projects. We contributed our data on KillerRed, applied to analyse its potential risks and the consequent measures taken to ensure the safety of the researchers and the general public, as well as preservation of the external environment (see below). We hope the collaboration with the ATOMS-Turkiye team will help encourage other teams to communicate about Safety; as it is as essential as the synthetic biology we work on, communication and collaboration should be on the same level for both. Thus we put this collaboration on he same level as synthetic biology for our team's outreach effort.

To analyse the risks present in our project and their possible consequences, we need to take into account all the parts that we bring together inside a single bacteria. This is done with the safety sheet. Here is a final version of our safety sheet as of 08/30/2013. The document is completed and signed.
In our project, KillerRed is the new part that we have to study in order to determine its potential effects. Before working with the actual DNA, documenting ourselves on the protein was necessary.
For safety considerations, several aspects of the protein have to be taken into account:

• The protein function
• The protein structure

In the case of KillerRed, the function is that of a fluorescent protein similar to EGFP or dsRed, but with photosensitizing properties increased 1000-fold. EGFP is used in many research applications as a reporter protein but is actually slightly toxic.[1,2]
This phototoxicity is due to ROS (Reactive Oxygen Species) generation. This ROS generation is only possible with the presence of molecular oxygen as an electron donor [3,5]. with superoxide being the main ROS generated, and the main source of KillerRed's phototoxicity [3]. ROS are extremely unstable compounds that readily react with many substrates including lipids, proteins and DNA [4]. There are many possible reactions and most if not all damage the substrate, leading to cell death. Reactions with DNA can also lead to mutagenesis and it was necessary to take this risk into account when working with the protein.
Since the protein generates ROS, the risk of researcher exposition to ROS is also present, for example if part of a KillerRed-expressing culture was spilled on an exposed body part.
Once the risks have been identified, scenarios can then be imagined in which one or more parts of our system are accidentally or intentionally released, leading to exposure of researchers, the external environment or the general population to biological material.

The first imaginable case is one where KillerRed-expressing bacteria come into contact with researchers and are unknowingly transported outside. These bacteria are resistant to ampicillin and produce a photosensitizing protein.
The bacteria themselves pose no particular risk for several reasons. First of all they are E. coli strain K12, widely used in laboratories and known to be in the BioSafety Level 1 group of micro-ogranisms. They are thus not known to cause any disease in humans or other organisms in the wild appart from allergic reactions. Their expression of KillerRed does not give them any advantage for survival in the wild: on the contrary the protein is toxic to the cells when they are illuminated. If they were spread in an environment away from light, there is no reason to think the protein has any other effect, based on its structural similarity with other fluorescent proteins such as EGFP, which are well-studied. As there is generally no presence of ampicillin or other beta-lactam-based antibiotics in the environment, the cells' resistance to ampicillin gives them no advantage.

The second case is where a solution containing KillerRed is spread outside or comes into contact with other organisms, for example a cell lysate in which the protein wasn't denaturated. In this case no genetic material is spread and so we focus on the chemical properties of KillerRed itself. KillerRed is a macromolecule (26.55 kDa [6]) and so cannot enter cells without specialized transporters. These transporters aren't present in E. coli, and probably aren't present in any other organism, since KillerRed is engineered and presents unique structural features which wouldn't be recognized by transporters for fluorescent proteins should they exist.
This leaves us to discuss the potential effects of KillerRed as a photosensitizer on the outside of cells, unlike clinical photosensitizers which readily penetrate cells. Due to the low lifetime of ROS, we only have to consider damage to membranes, in which case KillerRed could be considered a light irritant. Indeed, ROS reactions with membranes, primarily composed of lipids, leads to lipid peroxidation, which damages membranes and causes cell death. With the skin as a thick protective barrier compared to the scale of the protein and possible interactions of ROS with other molecules, the chemical risks posed by the protein itself are negligible both for humans and other multi-celled organisms alike. For microorganisms, the effect has not been tested, but it would take enormous mounts of the protein for it to be in a sufficient concentration to cause damage in the external environment. It is a stable molecule and doesn't degrade by itself quickly, but proteases in the external environment can easily destroy them.

As KillerRed by itself does not pose any risks in genetic or protein form, we now move on to the other components of our system: the Voigt light-regulated protein expression system.
As they confer no survival advantage nor have any effect other than regulating the expression of KillerRed in our system, these components do not pose a risk by themselves. The antibiotics used to maintain the corresponding plasmids in the cells have the same properties as ampicillin; resistance to them does not confer a survival advantage to bacteria in the wild. To prevent our bacteria ever making it inside a hospital where they could pose a risk due to the particular nature of those environments (immunodeficient organisms, strong trace presence of antibiotics...), we did not allow any sick researcher to perform experiments in the lab.

We show that interactions of our system with the environment or healthy organisms do not pose any more risk than interactions with wild-type bacteria. Yet this isn't all there is to biosecurity. As a general guideline for good laboratory practices, and by law, we are required to follow a set of rules. These are given by governmental organizations in France:

• The High Council for Biotechnologies
• The Ministry of Higher Education and Research
• The European Parliament

Among the laws that have passed concerning the use of genetically modified organisms, the following laws concern us in particular:

• European Directive 89/391/CEE
• European Directive 90/219/CEE

To sum up these different laws, we can make a list of good laboratory practices that we have to follow:

• 1. Minimalise contamination of work areas and the environment with genetically modified organisms.
• 2. Take measures to control risks at their source (genetic and/or protein level) and then use personal protection equipment (blouses, protectice eyewear...) if needed.
• 3. Regularly check and maintain equipment and procedures used to control genetically modified organism proliferation (laminar flow hoods, bleach...).
• 4. Verify if genetically modified organisms can thrive outside of their defined containment zones, and check for their presence in the work area out of their containment zones.
• 5. Ensure that personnel are adequately trained (with updated training for new techniques, risks and organisms in the lab).
• 6. Create groups or sub-groups dedicated to matters of biosecurity within the lab or work areas.
• 7. Design and publish good laboratory practices manuals for personnel working in the lab.
• 8. The lab has to notify the relevant authorities of the genetically modified organisms kept in it.
• 9. Lab personnel have to keep a record of their work on genetically modified organisms for tracability (the lab notebook!).
• 10. An emergency protocol has to be defined in the case of an emergency or accident involving the release of genetically modified organisms in the external environment, so that the health of researchers, the general public and the integrity of the environment can be protected.

The law also repeats all of iGEM's rules involving the BioSafety Level (BSL) of micro-organisms used during our projects:br>

• All chassis and used DNA sequence characteristics (function, known effects on humans and other organisms, origin...) have to be known and taken into account for risk evaluation.
• No pathogenic organism or known pathogenic DNA part may be used without special approval by the High Council for Biotechnologies

The European Directive 90/219/CEE also classifies BSL levels and the corresponding level of equipment and safety measures that need to be executed in order to limit or prevent dissemination of genetically modified organisms into the external environment. The document can be freely accessed here (in french). As there aren't only biological risks in a synthetic biology laboratory, we also need to take into account safety measures for the other risks. These are principally chemical risks relating to the use of certain chemicals in synthetic biology. These chemicals can be ethidium bromide (a known mutagen), guanidine hydrochloride (poisonous) or acrylamide (a known neurotoxin), among others present in the lab. The prevention measures put in place by the labor code in France include:

• 1. The evaluation of risks in the laboratory
• 2. Adapting the work and the workplace to the individual rather than the reverse
• 3. Continuously keep work areas up to date with recent equipment
• 4. Replace dangerous chemicals with less dangerous chemicals where possible
• 5. Plan risk prevention, taking into account techniques, workplace organization, work conditions and social relations between employees and possible external factors.
• 6. Prefer collective protection measures over individual versions.

In order to set an example for safety, we have strived to respect these rules all along our project and will communicate them to the next Grenoble iGEM team in 2014.

References

[1] Rouzbeh R. Taghizadeh and James L. Sherley, CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells, Journal of Biomedicine and Biotechnology, Volume 2008, Article ID 453590, 9 pages.
[2] Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ, Is green fluorescent protein toxic to the living cells?, Biochem Biophys Res Commun. 1999 Jul 14;260(3):712-7.
[3] Russell B. Vegh et al., Reactive oxygen species in photochemistry of the red fluorescent protein ‘‘Killer Red’’, Chem. Commun .,2011,47,4887–4889.
[4] Elisa Cabiscol, Jordi Tamarit, Joaquim Ros, Oxidative stress in bacteria and protein damage by reactive oxygen species, Internatl Microbiol, (2000) 3:3–8.
[5] Shawn Swavey and Matthew Tran (2013). Porphyrin and Phthalocyanine Photosensitizers as PDT Agents: A New Modality for the Treatment of Melanoma, Recent Advances in the Biology, Therapy and Management of Melanoma, Dr. Lester Davids (Ed.), ISBN: 978-953-51-0976-1, InTech, DOI: 10.5772/54940. Available from: http://www.intechopen.com/books/recent-advances-in-the-biology-therapy-and-management-of-melanoma/porphyrin-and-phthalocyanine-photosensitizers-as-pdt-agents-a-new-modality-for-the-treatment-of-mela
[6] According to bioinformatics.org's protein molecular weight calculator. [7] Directive 90/219/CEE of the European Council regarding the use of confined genetically modified organisms