Team:SydneyUni Australia/Safety

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Safety

The USyd iGEM team thoroughly considered the safety issues related to the project. This was important in order to ensure the health and wellbeing of the team members as they worked in the lab, as well as to protect the general public and the environment. The process encouraged us to think and act as researchers as we assessed the safety implications of our project to public health, security and environment in both the short and long term.

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

• researcher safety,
• public safety, or
• environmental safety?

Researcher safety

Inevitably there are risks associated when working in a laboratory and with using GMOs. However it was our responsibility to minimise these risks by taking appropriate precautions. We were designated space in a PC1 lab at the Biochemistry and Microbiology Building at the University of Sydney. However before we could commence any lab work all team members underwent a safety induction with our supervisor, who is also the Chair of the Safety Committee. We were shown the locations of fire extinguishers, emergency exits and eye wash sinks. Safety procedures and in particular the OH&S policies that would be relevant to our work were also outlined to us.

General lab practices were always followed. This included wearing proper PPE gear, washing hands before and after entering and leaving the lab and other aseptic work techniques. Work was always done under the guidance and supervision of our iGEM supervisor or in the presence of other members of trained staff. Further, the team and supervisor discussed safety issues whenever they arose. Only Escherichia coli strains were used in our labwork. These are categorised as Biosafety Level 1 organisms by WHO and considered exempt under the Australian Gene Technology Regulations ACT 2001. This means they are well-characterised and known to be non-pathogenic. As such, we ensured microbes were handled appropriately at all times. Immunocompromised persons were not permitted to work with these microbes until completely recovered. Bacterial cells were always disposed of in labelled bottles containing ethanol. All equipment used for growth or transfer of bacterial cells were decontaminated by autoclaving. Surfaces were sterilised before and after work was performed. Petri dishes and bottles containing bacteria were always labelled clearly, dated and incubated in appropriate locations.

There were also safety issues related to chemicals used and produced. In general, proper PPE gear was worn, chemicals were labelled and stored in a designated area of the lab and any spills were cleaned immediately and appropriately. Use of the following chemicals required particular care:

Chloroform and phenol are volatile chemicals, and if inhaled can cause lung edema and or complications to the central nervous system. It is also corrosive to the eyes and skin. The fumehood was used to prevent inhalation.

Sodium Hydroxide was always used wearing gloves and goggles to protect the skin and eyes.

Ethidium Bromide can irritate the lung and skin if exposed. It is also potentially carcinogenic. Gloves were worn when handling this.

Ethanol is volatile and highly flammable. They were therefore always stored in bottles with lids. Bunsen burners were kept at a safe distance away from equipment containing ethanol. Along with being an irritant or the eye and skin it also has intoxicating and severe dehydrating effect if ingested. To minimise accidental ingestion of ethanol no foods or drinks were allowed to be consumed in lab.

DCA (Dichloroethane) is a known carcinogen and its decomposition is the ultimate target of our project. As well as an irritant to the skin and eyes DCA can cause organ and nervous system damage upon long term exposure. Correct disposal of these chemical wastes needed to be ensured. This meant nothing was to be washed down the sink or placed into regular garbage bins. Proper PPE was worn and the fumehood was used.

Mercury thiocynate, used to perform a chloride assay is extremely hazardous if inhaled. It was strictly used in the fumehood only.

Acrylamide, used for the SDS-PAGE of protein extracts, is a known neurotoxin. It was used only at a specially designated workstation and proper PPE worn at all times.

Although national regulation regarding GMOs prohibits their release it was important to consider the effects our recombinant microbe may have on public health and the environment in case of accidental. Though, with good laboratory practices it was unlikely for our recombinant microbes to accidentally escape into public domain.

Public safety

Our host microbes cannot cause outbreaks in the general population. Genes for antibiotic resistance were used as selective markers. There is always the risk of antibiotic resistant populations arising from the misuse of antibiotics. Therefor proper disposal and decontaminating measure needed to be taken to prevent lab strains entering into the community.

Environmental safety

Our recombinant microbes are not intended for use in the environment to carry out its bioremediation activity at this stage in our project. We are attempting to build and characterise a pathway which could be used in future in E. coli or another chassis. If accidentally released it is unlikely that our microbe would survive, or that the genes we are characterising would spread through a population due to loss of the plasmid carrying them in non-selective conditions.

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

Toxicity or virulence None of the genes encode for virulence factors or toxins. This has been confirmed with literature and BLAST searches. Most of these metabolic enzymes are involved in detoxification.

Toxic intermediates While the enzymes themselves are not dangerous the products they form can be. Alcohol dehydrogenase reacts to give the intermediate product, chloroacetaldehyde. If it accumulates it is not only toxic to bacterial cells but may also put researchers at risk. It is categorised as a Group 1 carcinogen by WHO. Our construct has been designed to limit this from happening. The gene for acetaldehyde conversion has been placed directly after the gene responsible for its formation. This will mean the two enzymes are expressed almost simultaneously preventing build-up of the toxic product. dhlA and dhlB which encodes for dehalogenases release chloride atoms. A byproduct of this catabolic reaction is HCl. Proper PPE was worn to avoid contact with this and other products of these reactions.

Sequences

• The source organisms for the sequences of these genes are not known pathogens. Adh1b1 is derived from the human genome. While its use is in compliance with the Gene Technology Act some ethical concerns may arise in the general public.
• Our promoter, Psyn, was incorporated into our system. It is based on the lac promoter, which is obviously well characterised. However, Psyn is not inducible, therefore is constitutively expressed.
• The plasmid pSB1C3 was used as a vector for our device. It is non-conjugative and non-mobilisable. It therefore limits the potential for horizontal transfer of our parts.

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

An Institutional Biosafety Committee (IBC) exists at the University of Sydney. All projects involving genetically modified organisms (GMO) must be reported to and approved by the committee. The regulations are derived from the Australian Gene Technology Act 2001. Our project has been approved and complies with the IBC’s biosafety policies and regulations.

4. 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?

Safety Sheet