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From 2010.igem.org

The Perceived Dangers Associated with Gene Technology and Creating Artificial Life

With the rapid advances and the high impact of molecular biology in our lives, it is extremely important to be aware of the consequences science has on society and our environment. Scientists do most of their work in enclosed laboratories and following strict guidelines governed by regulatory bodies (in Australia the Office of the Gene Technology Regulator - OGTR). Though regulations dictate the containment of all genetically modified organisms (GMOs), there have been several cases in which GMO's have been unintentionally released.

For these reasons we were interested in collaborating with Penn State who undertook a survey on the perceptions of Synthetic Biology in society and the acceptance of GMO's in our everyday lives. Societal perceptions will continue to change as biotechnology increasing is rolled forward to deliver greater outcomes to mankind and such surveys are important benchmarks to these changes in perceptions and practices.

The RMIT 2010 iGEM team is attempting to create a biological system that will produce therapeutic peptides at a low economic cost. A biological machine will be created that uses an inactivated thermostable polymerase as a carrier protein with an attached desired peptide - in our case a therapeutic peptide. There are several safety concerns when undergoing this project that we have addressed:

Our project involves working with polymerases, which are proteins found in all living organisms and are involved in the process of DNA replication. More specifically Taq polymerase (found in the Archeon Thermus aquaticus), being a revolutionary enzyme in the field of molecular biology due to its thermostability, was the focus of our work. The immediate danger is a bacterium containing a Taq plasmid part escaping into the environment and altering the genetic material of many organisms. RMIT team has reduced these dangers by creating mutations that will not allow this protein to bind to DNA or undertake polymerase activity.

The chosen peptides that are being ligated with the Taq polymerase are Losartan and arginine vasopressin. Losartan is a peptide that reduces blood pressure as an antagonist of the angiotensin system. This peptide is safe if not ingested intentionally for harmful purposes as recognised by drug and therapeutic agencies world wide.

The second peptide is arginine vasopressin (AVP), commonly known as antidiuretic hormone. It is associated with water, glucose and salt regulation in the human body and, unlike Losartan, it increases blood pressure.

The bacteria used are Escherichia coli XL-1-Blue and NEB Turbo (High Efficiency), which are commercially available E.coli strain derived from E.coli K12 and used for routine and widespread molecular biology studies. Derivatives of K12 are avirulent and may be considered to be ACDP biological agents hazard group 1 because they have an established record of safety in the laboratory with no adverse effects on human, animal or plant health or the environment. Furthermore E.coli XL-1-Blue is incapable of survival outside the laboratory.

Safety Issues

1. Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety?

No facet of our project would cause significant safety issues. All work is to be contained in PC2 labs. Processes carried out will be done so only after training is received and will be supervised by a skilled instructor to ensure the proper protocols are followed. Our labs will also diminish risks by avoiding the use of carcinogenic products like ethidium bromide, and instead use a more safe nucleic acid stain. This stain – GelStar - does not require UV light to be visualized but blue light, making it safer. The environment will be protected from contamination by waste products because any dangerous material will be disposed of in the correct container (e.g. biohazard containers for biological waste such as E. coli colonies), autoclaved and disposed of responsibly by the university. Team members will also be taught proper molecular biology skills and aseptic techniques. Public safety is ensured as no member of the public is permitted access within the PC2 labs unless approved by the university. Team members will follow proper PCR facility procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, o Did you document these issues in the Registry? o How did you manage to handle the safety issue? o How could other teams learn from your experience?

None of our newly engineered BioBrick parts raise safety issues. On the contrary, our system may actually help in reducing risk overall because if the platform works it will reduce the usage of chemicals associated with protein purification.

As previously stated as a safety measure the Taq polymerase part has been mutated in such a way that it will no longer bind to DNA. Through these mutations we eliminate its function and will just be a harmless globular protein in the cell. According to the MSDS, Taq polymerase does not produce any toxic compounds but may do so in the presence of strong oxidising agents/bases, degrade to form CO and CO2. In such an event, this side effect can be combated simply by ventilating the area.

If a biopart, for example the modified Taq polymerase, mutated back to bind to E. coli DNA it might kill the cell. This however is unlikely and in any case would only result in the destruction of some E. coli.

3. Is there a local biosafety group, committee, or review board at your institution?

Yes. Our supervisor, Dr. Leonard Pattenden is on the biosafety committee. There was an internal requirement to complete a project risk assessment to cover all areas of the project. Our project has been deemed low risk. Additionally, our project required registration for a “dealing” under Australian regulations from the Office of the Gene Technology Regulator (OGTR).

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Ideally, risk assessments should be conducted for each individual depending on their experience and expertise in handling the chemicals/organisms and carrying out the protocols. In addition, each student should write a risk assessment of the protocols employed to complete the project. This way all students will know and understand the risks of each experiment being done.

Knowledge is power: the more information known about a newly created part/device/system, the easier it is to identify and combat the associated risks.

5. Could there be an unplanned event or series of events involving your project, resulting in either death, injury, occupational illness, death, damage to equipment or property, or damage to the environment?

Any time a person steps into a laboratory there is a risk of a serious emergency occurring.. The likelihood of such an occurrence has been reduced due to the risk management techniques employed in the laboratory plus the oversight and training of students to comply with OHS best practice standards. It’s important to note however that risk assessments focus on the danger of the task as opposed to the skill level of the person carrying out the task (e.g. a postgraduate is less likely to injure themselves than an undergraduate). This means risk assessments on students need to be suitably adjusted.

6. Does your project require the exposure or release of the engineered organism to people or the environment (e.g. as medicine, for bioremediation)?

This project involves the creation of new technology to produce drugs at a large scale with high purity with a significantly lower cost. If the experiment is successful, the next step in this experiment would be testing the effect of these drugs on animal models. The bacterium itself would not be released, but the product created by it would.