Given the nature of our project we were aware that it could carry potential risks and were mindful of these throughout our time developing, carrying out experiments and when thinking about project applications.
Safety and Our Project
Use of Organisms
Aphids (Myzus persicae)
We originally planned to carry out experiments which would have involved the use of aphids. We were aware that these were not on the iGEM White List and therefore had to complete a Check-In form. The risks that the aphids carried included the issue of keeping them in a contained yet suitable environment, ensuring their well-being but also preventing them from escaping. However, since the aphids themselves were not transgenic and are native to local ecology this would not have had any significant effect on the environment.
Tobacco Plants (Nicotiana benthamiana)
Throughout our project we used Nicotiana benthamiana as the expression system for our genetic constructs. This meant infiltrating them with engineered Agrobacterium tumefaciens to produce transgenic plants. The plants themselves pose no risk to the environment or to humans, even once we created transgenic plants, since Agrobacterium has been shown to not have any negative effects on humans. That said, the plants were always kept contained within the lab and were disposed of properly when finished with.
We used E. coli as a chassis organism to create the genetic constructs for our project. The strain of E. coli we used (DH5 alpha) during the project is non-virulent and therefore poses no immediate risk to humans or the environment. Although E. coli can give rise to a range of diseases as an opportunistic pathogen, we took care that contact with it was prevented by wearing lab coats, goggles and gloves when working with it, as well as ensuring thorough hand washing and cleanliness throughout.
We also used Agrobacterium (strain GV3101) as a chassis organism to transform Nicotiana benthamiana. As with E. coli it is an opportunistic pathogen which can give rise to some diseases, however we prevented contact and spreading of it outside of the lab by wearing lab coats, goggles and gloves when working with it, as well as ensuring thorough hand washing and cleanliness throughout.
Experimental Safety Risks
Our project has required a range of experimental procedures which have all carried risks which we have done our best to prevent. Here we have listed the hazards that we were aware of and took precautions to overcome during our time in the lab.
Taking care when carrying out PCR and gel electrophoresis which we used to test colonies positive for constructs and for the detection of siRNA constructs derived from transgenic plants. Hazards here included using razor blades for the excision of DNA out of agarose gels and the use of transilluminators to visualise gel electrophoresis products which poses a risk due to UV.
Throughout our project we required the use of liquid nitrogen for the creation of competent cells, Agrobacterium transformation and RNA extraction from transformed plants.
The use of Agrobacterium posed a small risk since it is possible that it could transfer DNA to the environment as it is a plant pathogen. We took precautions to minimise this risk such as keeping the Agrobacterium work contained in the lab, wearing lab coats, goggles and gloves when handling it, and disposing of it correctly when finished.
The use of aphids themselves also posed a small safety risk. They are plant pests but are native to our local environment and if they had escaped would pose a low risk on the environment. Also, the aphids were not transgenic and so if they had escaped would carry a low risk to our immediate surroundings. We were also specifically targeting aphid genes with RNAi which is not transgenerational and carries very low risk if it were to leave the lab. Precautions we took to minimise this risk included wearing lab coats, goggles and gloves when working with it.
Laboratory Safety Risks
Prior to starting the lab work for our project in our university laboratory, all our team received a safety induction talk from the head of the lab. During this induction we covered a range of topics including emergency procedures (fire and first aid) and general laboratory health and safety. We were then required to read and sign several risk assessment and code of conduct forms that are appropriate for general lab safety and for the use of genetically modified organisms. The laboratory staff and our project leaders were fully aware of the experiments that would be performed during our time working within the lab. This ensured that our project, and how we worked, obeyed the Cardiff University laboratory safety guidelines but also local, national and international lab safety standards, which included the use of genetically modified organisms. Our lab was at a biosafety Level 1, meaning that all work carried out was low risk.
Throughout the project we took care to ensure that these rules and regulations were abided by via appropriate lab behaviour and following appropriate procedures for the manipulation of genetically engineered organisms. One specific issue covered in our risk assessment was the escape of the aphids which we controlled in a specialised plastic container designed with airtight doors but wire mesh on the sides to ensure that air could pass through, but aphids could not escape. The local risk assessment involved identifying where the aphids could stay in the greenhouse, an investigation into the security of the container, and analysis of any potential risks of aphid escape. As these aphids are native to the local area and not transgenic, they pose no risks.
Safety and Project Application
The aim for our project was to develop a product that could be applied in the agricultural industry as a biological pesticide. However, with this we realised that there would be safety risks involved.
For example, we ensured that our RNAi constructs were specific to aphid genes and that they have no off-target effects, particularly on humans. After speaking to a representative from the Gwent Bee Keepers at one of our outreach events, it was brought to our attention that our product may have detrimental effects on honey bees and so we contacted the Welsh Bee Keeping Association (WBKA) for further information on this. To establish the effects our project would have on the environment bioinformatic analysis was carried out. We found that there were relatively low hits on honeybees and humans, with the majority of hits on Myzus persicae. With this in mind we were happy that our application would specifically target aphids as crop pests with minimal off target effects.
Another concern raised was that the use of genetically modified plants, as this topic can be a controversial subject for the public. We carried out a survey as part of our human practices which aimed to gather information on the public’s perspective of the use of GM and integrated this in our human outreach to minimise concerns and create discussion around the topic. This approach has been received well by the public at the outreach events we have attended.
Finally, the bacterial species involved in our project were raised as a safety concern. However, these strains have been used in the past and are common in experimental procedures of this nature. We are happy that they would pose no safety or security issues as they would be handled and worked with in specialised, highly sterile and controlled environments.