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When working with proteins like KillerRed, one can quickly raise safety issues. We have been contacted by the FIXME 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.
First of all, 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 include link to KR documentation page here ourselves on the protein was necessary.
For safety considerations, several aspects of the protein have to be taken into account:
In the case of KillerRed, the function is that of a fluorescent protein similar to GFP or dsRed, but with photosensitizing properties increased 1000-fold. GFP 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.
Next episode coming soon!
[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
