Team:Imperial College London/Safety

The safety standards in Imperial College are very high, but in order to do high-end research some dangerous procedures cannot be avoided. With regard to our project those were in particular the use for toxic chemicals, including:

• [http://www.sciencelab.com/msds.php?msdsId=9927131 Chloramphenicol] is a broad-spectrum bacteriostatic antibiotic which is effective against many Gram-positive and Gram-negative bacteria, including most anaerobic organisms. As a result of resistance as well as health risks, the use of chloramphenicol is limited to topically treatment of eye infections. Side effects of the antibiotic may include bone marrow toxicity: bone marrow suppression defined as a direct toxic effect of the drug which is usually reversible, and aplastic anaemia, which is idiosyncratic and generally fatal.

• [http://www.jtbaker.com/msds/englishhtml/p7896.htm Catechol] (also pyrocatechol or 1,2-dihydroxybenzene) is an organic molecule with the formula C6H4(OH)2. Classed as ortho isomer it is found as three isomeric benzenediols. This colourless compound occurs naturally in trace amounts but about 20’000 tons are produced annually, mainly as a precursor to pesticides, flavours and fragrances. It is classed as an irritant in the [http://ec.europa.eu/environment/chemicals/dansub/home_en.htm EU] but classified as Toxic in the USA [http://www.cdc.gov/niosh/ipcsneng/neng0411.html] [http://www.cdc.gov/niosh/npg/npgd0109.html] and has furthermore been declared a possible carcinogen.

• [http://www.biotium.com/product/product_info/MSDS/msds%2041002.pdf GelRed™], and [http://tools.invitrogen.com/content/sfs/msds/S33110_MTR-EUIV_EN.pdf SYBR®Safe] were used frequently to stain DNA in gel electrophoresis rather than [http://www.jtbaker.com/msds/englishhtml/e2410.htm ethidium bromide] which is much more toxic as well as being a powerful carcinogen and was therefore completely eliminated from use in our lab.

Chemicals that have been demonstrated to be toxic or are classed as irritants were not handled by the team at critical concentrations, but rather diluted for us by more experienced members of staff, like our supervisors. This minimised the risk to the all people working in the lab and ensured correct safety practices were taken during the most precarious steps. These substances were stored in locked cupboards and handed to us diluted in aliquots of constant volumes.

• In order to make stained DNA visible UV light is normally used. This can lead to UV exposure especially when DNA bands are excised from the gel. For this reason a blue light box and safety goggles with light filters were used for these steps to prevent damage to the retina as well as our skin.

Generally good laboratory practices were adhered to, including the wearing lab coats and protective gloves when in the laboratory. Furthermore all waste, including live bacteria and toxics, were disposed of by trained members of staff according to sound safety protocols.

Public Safety

Of course problematic ideas were developed during the course of our project, however those were eliminated in our finalized project description. In order to minimize the risk our project poses to public safety several precaution steps were taken:

• Application of our product: During the design phase of our product much thought went into a safe way to use a GMO as biosensor. We quickly decided to design our project in a way that would avoid both release of the organism and exposure of humans to the bacteria by restricting the bacteria to small containers to which samples are added.

• Choice of chassis: We deliberately chose a non-pathogenic bacterium – B. subtilis – to serve as our chassis, as it is much less likely to cause harm to exposed people than E. coli for example.

• Choice of genes: As laid out early by our human practices work show, we aimed to minimise the use of genes from pathogenic organisms. A good example for how we did this was rejecting the use of the sortase A – important for attachment of virulence factors to the cell wall – of Staphylococcus aureus to anchor our cell wall binding protein, and instead search for an alternative such as LytC from B. subtilis which plays no role in virulence but fulfils an equivalent role. If there was no alternative to genes from pathogenic bacteria, such as the ComCDE system of S. pneumonia, we checked for homologous systems in non-pathogenic systems and researched the role the system could play in virulence. Only if no link to pathogenicity was found, like in the case of ComCDE, we considered using the gene in our project.

Overall this system should not be able to cause any harm to the public under any foreseeable circumstances. Our project idea was modified several times during its development in order to fulfil all the safety guide lines we had agreed on in our human practices work shop as well as our panel discussion with experts from many fields, including Synthetic Biology and Ethics.

Environmental Safety

During the development of our project we put a lot of though into the application of our biosensor and how people in LDCs could benefit from it. During this process we came to the conclusion that in order to minimise the environmental risks of our project no bacteria were to be released into the environment but rather that water sample should be take and out into a container in which our bacteria were grown. Environmental safety was inherent in our design so that no risk should arise of our project, as we will demonstrate in detain in the last section of this page were we propose new safety standards for products of Synthetic Biology to make them more environmentally friendly.

None of our biobricks poses as safety issue on its own. However our output system, which makes use of the protein XylE must be handled with care. XylE itself is perfectly harmless however the substrate it acts on – Catechol – is classed as an irritant in the EU and as toxic in the USA (see above). As pointed out in our Protocols as well as the Registry this means that when making use of our output system or other biobricks containing XylE to create a visual output, this chemical has to be used carefully.

One of the modules contains the genes ComC, ComD and ComE which are native to S. pneumonia. However the system was only handled in fragments and was synthesised for us by an europhins|MWG so genomic DNA of pathogenic organisms was not used at any stage of our assembly process. Furthermore, as mentioned before, the ComCDE genes do not play a role in virulence but are instead involved in cell competence, so no risk arises to us, or people using these genes or our system later.

How did we manage to handle the safety issue?

As described in the first section of this page, toxic substances like catechol were handled by our supervisors at critical concentrations and only used by the students when sufficiently diluted.

How could other teams learn from our experience?

As mentioned the risk assessment of catechol varies between the EU and the USA. As a result we can recommend other teams to check unfamiliar substances across several regulatory bodies in order to make a more informed assessment of the risks associated with a compound. Catechol in particular is also suspected to be a carcinogen, as a result of which our supervisors and us handled it with even greater care.

Prior to the commencement of our projects the following had to be undertaken:

• an adequate risk assessment.
• submission of this risk assessment form to the local Genetic Modification Safety Committee and approval of both the risk assessment and identified control measures by that committee.
• the implementation of such control measures.
• if our project had been Class 2 or higher, the project would have been discussed with the Director of Occupational Health to determine whether health surveillance is appropriate.

The risk assessment form included information about all people involved in the project but also listed all strains of microorganisms as well as the genes we used and their function. Following the consideration of the iGEM project by the local GM Safety Committee, the Chair of this committee signed the GM risk assessment form and issued a green approval form detailing the requirement to notify the work to the HSE to the Principal Investigator (PI) for the project.

All students a working in the life sciences department of Imperial College have been given a lecture on safety procedures to be followed in our labs and all members of the iGEM team were given an additional lab induction by one of our supervisors.

Disruption of essential genes: We designed our vectors so they would integrate into the B. subtilis genome and interrupt gene sequences involved in starch synthesis (amyE) and uracil synthesis. As a result, the final version of our product would only be able to survive in special conditions where these additional nutrients are provided. Therefore, if it were to be released into the environment, it would in theory only survive for a limited period of time. Furthermore the lab strains of B. subtilis cannot synthesis tryptophan, as a result of a gene knock-out. Therefore the probability of any of the released bacteria acquiring all three different gene sequences, which are essential for its survival, by horizontal gene transfer is vanishingly small.

Removal of antibiotic resistance: One major public concern regarding the safety GMOs is the potentially wide spread introduction of antibiotic resistance into the environment and the consequences for treatment of individuals infected with multi-drug resistant bacterial strains. The resistance is important in the laboratory setting for selection of transformed bacteria so a compromise had to be found. We have used a recombinase system native to B. subtilis that will allow us to excise the resistance genes after the selection process as these are between target sites of the recombinase. One of the systems that can be used to achieve this is the Dif system.

Other safety mechanism: An additional level of confidence is that catechol, which is a substrate needed by XylE to give a colour output, actually kills the bacteria a few hours after it is added. Nevertheless, we are aware that in order for this to occur, the catechol must be added appropriately, and this relies on the correct use of the detection kit. However, if the bacteria were released into the environment without being exposed to catechol, the original genome disruption would be sufficient to ensure that the bacteria would not survive.

However we recognise that biological systems are inherently unpredictable and have the capacity to change. Nevertheless, we believe that the benefits of controlling the population and spread of Schistosoma makes this an important project to pursue on the proviso that biosafety issues and environmental impacts are closely monitored, controlled and managed. We advocate continued, regular testing of the bacteria in order to ensure that any mutations, which would result in the deviation from its function, are noticed early and so the problem can be remedied. We would ensure that there are thorough, controlled and longitudinal studies of the effects of the bacteria on a range of environments and seasons, prior to the marketing of the product.