Are there any safety issues in the lab
or for the wider public and environment?

Safety issues have been considered throughout our project design, experimental work plan and thorough risk assessment to achieve quality by design. Furthermore, all the participants of the project have completed a safety induction given by the Departmental Biological Safety Officer, during which fire evacuation procedure, accident reporting and general safety practices have been detailed and all team members have a copy of the departmental safety book. E. coli strains that are non-pathogenic and therefore low-risk have been chosen, so there is no great safety issue with regards to the host organism as long as Safe Microbiological Technique (SMicT) is adhered to. The major hazard identified in our project is the use of Ethidium Bromide (EtBr) for staining of agarose gels. EtBr is a mutagen and moderately toxic after an acute exposure due to its ability to intercalate with DNA bases, causing mutations such as “frameshift” and deletion, leading to oncogenic development. It should be treated as a possible carcinogen and teratogen. The following control measures will be adopted:

• Nitrile gloves will be used for all EtBr handling
• EtBr preparations are to be carried out in a dedicated fume hood
• All EtBr-containing materials will be disposed of in dedicated bins
• Work surfaces must be thoroughly cleaned after each experiment

In addition, attention should be paid when operating the centrifuge, autoclave as well as microwave. For instance, weight distribution should be even in the centrifuge to prevent the rotor blade from dislodging and shredding the machine. Also, heatproof gloves should be worn when handling the autoclave to prevent first to second degree burns. Last but not least, when melting agar we must make sure the bottle cap is loosened to prevent pressure buildup.

Our project poses some risk in terms of public and environmental safety. As mentioned before, the organism we are going to use is a non-pathogenic lab strain of E. coli. However, one paper suggested that highly supercoiled plasmids which is our final product will enable a higher transfection efficiency. The potential environmental damage which may occur is the higher probability of a successful unintended transfer of antibiotic resistant genes to other bacteria in the ecosystem, thus potentially resulting in the development of multiresistant pathogens^[1]. Henceforth, proper chemical waste disposal and Genetically Modified Organism containment are essential. There is also a possible risk to security through malicious misuse by individuals, groups or states. Whilst our strain of E. coli is non pathogenic, the supercoiled plasmids produced from it can be used as a disease inducing vector. Precaution steps include:

• Liquids containing E. coli, other living organisms and equipment with biological contamination must be autoclaved before disposal
• EtBr waste and contaminated materials (gloves, tissues, etc.) must be put in the dedicated bin for collection by Estates. Work surfaces must be thoroughly cleaned after each experiment.