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 disposal of sharps, biohazardous material and trace chemicals. The team were also trained in the relevant fire safety procedures; the locations of fire blankets, fire exits, etc. 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, Standard Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles, masks and ear defenders were used when needed (e.g. for SDS-PAGE and exposure to UV light source, while sonicating cells etc.). While working in the lab, we were supervised by our instructors, advisors or lab technicians from Dundee University’s College 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 in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, Ethidium bromide is considered to be a mutagen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining for agarose gel electrophoresis instead.

As a safety precaution, all harmful chemicals were used within the sterile environment of a fume cupboard and upon contamination were disposed of promptly and correctly according to the relevant Material Safety Data Sheet (MSDS).

Biosafety

We used safe bacteria types in the lab: E. coli and Bacillus subtilis. E. 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, B. 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. B. 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, MC1061, JM110 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 little hazard to human health. The B. subtilis strains which we used were NRS3086 strain (derived from the 1086 strain) and 3610.

Although the bacterial strains we used are non-pathogenic, 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. 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. Reusable containers that had been in contact with live cells were soaked in Virkon solution to be disinfected before disposal and to ensure that no live cultures were poured into the sinks.

Given that our project’s primary concern is the toxin microcystin, we made sure to be very safe regarding its use. The volumes at which we used microcystin for experiments were at levels so low they could present no hazard to human health.

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 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 lacks the ability to produce large quantities of toxins that affect humans. The B. subtilis strains used were of minimal danger to the public as well - this organism has never been associated with human infection and is in fact used as a food source in Japan (Netto).

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.

iii. Environmental Safety

The use of genetically modified organisms in the environment is a contentious issue, however regardless of your opinion it is obvious that they must be kept isolated from the wild in case they harm the environment they have been engineered to protect, improve or enhance.

Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment. Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag – a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be mopped up by PP1 expressed by the bacteria.

Question 2: Are any parts or devices in our project associated with (or known to cause):

• i. Pathogenicity, infectivity, or toxicity?
• ii. Threats to environmental quality?
• iii. Security Concerns?

i. Pathogenicity, infectivity, or toxicity

The strains we used, which were derived from E. coli K12 lack many of the virulence factors required for infection. B. subtilis has never been associated with human infection. It has GRAS status which means it is “generally recognized as safe”.

ii. Threats to environmental quality

Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment.

Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag – a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be mopped up by PP1 expressed by the bacteria.

iii. Security concerns

Due to the non-pathogenic, non-toxicogenic, and non-colonising nature of E. coli K12 and B. subtilis strains which we utilised and the harmless nature of our parts, we do not foresee any security concerns with our project. Our laboratory has secure entry to prevent unauthorised access. In the wrong hands, nothing harmful could be done with our parts or bacterial strains.

Question 3: Do any of the new BioBricks parts (or devices) that you made this year raise any safety issues?

Our biobricks do not raise any direct safety concerns. Regarding our mop device and microcystin detector containing bacteria, if they burst in the environment for any reason, they might spread lots of the toxin back into the water. Although, what binds to microcystin? – PP1, so if the cells burst, surely the toxin would bind to the protein phosphatase 1 in the water!

We have considered how we might safely deploy the ToxiMop in the environment with the use of what we call the ‘ToxiTeabag’. A perforated bag with holes too large for bacteria to fit through but not water molecules, or more importantly, microcystin.