Team:Dundee/Safety

Question 1: Would any of your project idea raise safety issues in terms of:

• i. Research Safety
• ii. Public Safety
• iii. Environmental Safety

i. Research Safety

General

We all attended a general health and safety induction at the beginning of our iGEM project and were given a safety tour of our lab. The tour included guidance with regards to waste disposal of sharps, biohazardous material and trace chemicals. The team members wore disposable gloves and lab coats at all times when working in the wet lab and eliminated risk of contamination spreading outside the laboratory environment by ensuring they removed these items upon exit. Good laboratory practice, such as regular hand-washing and frequent cleaning of workbenches was enforced.

At all times, Safety Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles and masks were used when needed (e.g. for SDS-PAGE and exposure to UV light source). While working in the lab, we were supervised by our instructors, advisors or lab technicians from the Dundee University’s School of Life Sciences Learning & Teaching staff to ensure that we were safely carrying out procedures.

Chemical

To reduce risk to our health, we decided to use Qiagen kits (mini-prepping, gel extraction, PCR purification, etc.) rather than phenol based protocols. Ethidium bromide (EtBr) is an intercalating agent (it inserts itself into the DNA helix, unravelling the structure) commonly used as a fluorescent tag (nucleic acid staining) in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, ethidium bromide is considered to be a mutagen and carcinogen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining method for agarose gel electrophoresis instead.

Biosafety

We used safe bacteria types in the lab: E. coli and and B. subtilis. Escherichia coli is a Gram-negative bacterium which is naturally found in the colon of warm-blooded organisms such as humans. Some strains of E. coli are the cause of serious food poisoning in humans, but the majority are harmless. On the other hand, Bacillus subtilis is a Gram-positive bacterium and has the ability to form a tough, protective endospore. This allows the B. subtilis to tolerate extreme environmental conditions. Bacillus subtilis inhabits the human gut, and is thought to be a natural gut commensal.

We used a few different bacterial strains throughout our project: E. coli MG1655, JM110, 1061 and DH5α. These are all disabled, non-pathogenic, non-toxicogenic, non-colonising, laboratory-adapted K12 strains, which are widely used for research purposes and present absolutely no hazard to human health.

Although the bacterial strains we used are non-pathogenic (and therefore not labelled as a biohazard as such), it is still important that we took measures to prevent any contamination. Any protocols which involved the transfer of bacterial cells or bacterial colonies between plates or tubes were carried out in sterile conditions; close to a Bunsen flame. Otherwise, bacterial cells were stored in lidded containers/universal tubes with the caps sealed tightly. All of the biological waste was disposed of in accordance to lab waste disposal protocols, which involves autoclaving biological waste prior to discarding it. Re-usable containers that had been in contact cells were soaked in Virkon solution to be disinfected.

ii. Public Safety

While carrying out our project, we took utmost care to ensure that neither biological nor chemical materials were released from our lab accidentally. However, if unintentional release were to occur, the bacterial strains that we used would pose very little to no danger to human health. With regards to the E.coli K12 strain derivatives which we used, the lack of danger to people is due to its poor ability to colonize the gut and establish infections. E. coli K12 also appears to lack the ability to produce large quantities of toxins that affect humans. The B. subtilis strains used were of minute danger to the public as well.

We used ampicillin resistant genes within our plasmids as a selectable marker for bacterial transformations. As we are fully aware of the issues surrounding horizontal gene transfer and multi-drug resistant bacteria, we followed university protocols regarding GMO waste disposal. The ultimate goal of our project, as with any other iGEM project, is for our modified bacteria to be used practically in the environment to treat algal blooms by removing microcystin. Our final step would be to remove antibiotic resistance from our plasmid prior to release of our bacteria into the environment.