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

1. researcher safety,

2. public safety, or

3. environmental safety

Researcher Safety:

During the project the risk to the researcher was minimised in a number of ways. Standard lab protection was always worn (lab coat and gloves) and we adhered to COSHH forms. All team members involved in lab work attended a 2 hour safety briefing with the Safety and Risk Management Officer for the MIB (Dr. Tanya Aspinall). No eating, drinking or chewing was allowed in the lab. No shorts, skirts or dresses were allowed when in the lab. UV protective visors, gloves and lab coats were worn when visualising EtBr stained agarose gels under UV light. The lab we worked in is Biosafety Level 1 according to WHO Laboratory Safety Manual.

Organisms we used:

E. coli BL21(DE3) - Standard lab strain. Used Good Microbiological Practice (GMP) when handling.

E. coli DH5-alpha - Another standard lab strain. Used GMP when handling.

Synechocystis sp. PCC 6803 - Sequences for the delta-9 and delta-12 desaturase genes were taken from here and then synthesised - we never had any contact with the strain. This cyanobacteria is found in freshwater and is not thought to be a threat.

Chemicals used:

Ethidium Bromide: Hazard statements H302-H330-H341 - Harmful if swallowed, Fatal if inhaled, Suggested of causing genetic defects. Personal protective clothing must be worn. Used fumehood when handling. Gel containing ethidium bromide was disposed of in the hazardous waste gel box in the visualiser room. All gel kits that were used in conjunction with EtBr were kept together in a space dedicated to them in the lab. Pipette tips used with EtBr were disposed of in a designated EtBr pipette tip bin in the lab.

Chloroform: Hazard statements H302-H315-H351-H373 - Harmful if swallowed, Causes skin irritation, Suspected of causing cancer, Causes damage to organs through prolonged or repeated exposure. Personal protective clothing must be worn. Used fumehood when handling. Waste containing chloroform was disposed of in halogenated waste containers.

Methanol: Hazard statements H225-H301-H311-H331-H370 - Highly flammable liquid and vapour, Toxic if swallowed, Toxic in contact with skin, Toxic if inhaled, Causes damage to organs. Personal protective clothing must be worn. Used fumehood when handling. Waste was disposed of in organic waste containers.

Liquid Nitrogen: Hazard statements H281 - Contains refrigerated gas; may cause cryogenic burns or injury. Personal protective clothing must be worn, cold-proof gloves must be worn to avoid cryogenic burns.

Public and Environmental Safety:

GMP was also used in order to ensure public safety. Once something had come into contact with the bacteria we used, it was sterilised. Pipette tips were put into Virkon solution and then disposed of in Autoclave bags, ready for incineration. Flasks previously containing bacteria cultures were soaked in Virkon solution before being washed in the sink. As well as this, the bacteria were cultured so as to not be viable outside of a laboratory setting (due to the specific media they were grown on). This ensures that, even if living bacteria did manage to escape the lab, it would not have survived.

The same procedures as above were also used to ensure environmental safety. In addition to this, organic solvents that are potentially dangerous to aquatic organisms were disposed of in a separate waste container and removed professionally.

In the unlikely event that our re-engineered strains escaped our workspace, it is thought that no risk would be posed to the public or to the environment. This is because the bacteria we use is non-pathogenic and also would be at a disadvantage in any environment other than that found within the culture medium it is grown on. This disadvantage would be expected to kill the bacteria should they ever find their way into the outside environment.

Future safety implications if project was commercialised:

Once the methods have been sufficiently optimised, our project would have commercial potential. This would inevitably lead to further safety concerns and questions. Due to the wide range of products that the four main components of palm oil are used in, each application would have to be considered separately. For example, the use of synthetic oleic acid in cooking oils would raise concerns over whether or not this product would be safe for consumption.

In order to minimise any risk posed by using genetically altered E. coli, a number of steps would be put in place. Firstly, the vats containing the bacteria would be kept in a secure facility, with all workers fully trained in the handling of biological samples. This would ensure that the workers/researchers were not at any risk.

The chemical extraction of fatty acids from the E. coli cultures would theoretically separate all bacteria from the final product. However, at the risk of some bacteria remaining within the extractant, further methods would have to be put in place. In the case of palmitic and stearic acid, the samples would have to be separated from each other using fractional distillation. This technique involves heating the samples up and separating the compounds based on their boiling point. The high temperatures involved would kill any bacteria present in the samples. A similar technique could also be used in the case of oleic and linoleic acid too. The samples of these fatty acids could be refluxed, which would kill any bacteria present whilst keeping the sample in a sealed vessel. After these procedures have been completed, no viable bacteria would be present within the fatty acid products, and the public would not come into contact with genetically modified bacteria.

Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?

No, none of the BioBrick parts used this year are thought to be hazardous - they are all part of either the natural biosynthesis pathway of E. coli or were synthesised using sequences from Synechocystis sp. PCC6803, a cyanobacterium found in freshwater.

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

The University of Manchester has a university-wide Biological Safety Committee, whose guidelines for safe biological laboratory practices we adhered to throughout the project. (Guidelines can be found here)

A representative for the biosafety committee of the University of Manchester (Dr. Tanya Aspinall, Safety and Risk Assessment Officer of Manchester Institute of Biotechnology) gave the team a 2 hour talk about safety in the laboratory. During this safety training, the team was advised on how to handle potentially hazardous substances, such as liquid nitrogen, and how to safely dispose of certain materials. The team’s lab benches and experimental plans were also assessed and deemed safe to proceed with. Supervisors to the team were always on hand in the lab to make sure the team were working safely.

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?

To encourage safe laboratory practices, we believe it would be a good idea for completed generic COSHH/Risk Assessment forms for common procedures such as gel electrophoresis and PCR be available on the iGEM website. Having a collection of completed forms covering the most common protocols used during the iGEM competition would first ensure that teams carry out these procedures adhering to the iGEM Safety Committee’s standards, and secondly would give teams a guideline when filling out safety forms for non-standard procedures.

Safety forms were approved on September 22, 2013 by Evan Appleton.