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 PC2 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 [http://www.comlaw.gov.au/Details/F2011C00732 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.

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. However, 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. Therefore 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. The School of Molecular Bioscience also has a set of well-documented [http://sydney.edu.au/science/molecular_bioscience/ohs/ standard operating procedures.]


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?

Worker safety is a particular concern of ours. It is only through the harm to workers exposed to DCA that people began to care about DCA elsewhere in the environment. Furthermore, if one considers examples from the history of science, it is often researchers in new fields, with new technologies, that are at risk.

We have created a series of safety sheets to accompany all the protocols used in our project.

Protocol: Acetaldehyde Inhibition Assay
Name Risk Required Protection Equipment First Aid Response Storage
Acetaldehyde Flammable. Irritant to skin and eyes. Potential carcinogen. Goggles, gloves. Flush eyes with water. Skin needs to be washed with non-abrasive soap. Spills must be wiped using inert material. Dispose of material in appropriate contaminated waste. Water must be spread over contaminated surface. Store in tight containers away from light.


Protocol: Agarose Gel Electrophoresis
Name Risk Required Protection Equipment First Aid Response Storage
Ethidium Bromide Carcinogen. Mutagen. Slightly irritating to skin and eyes. Can be seriously hazardous if ingested Goggles, gloves. Flush eyes with water. Skin needs to be washed with non-abrasive soap. Spills must be wiped using inert material. Dispose of material in appropriate contaminated waste. Water must be spread over contaminated surface. Store in tight containers away from light.


Protocol: Chloride Assay
Name Risk Required Protection Equipment First Aid Response Storage
KP Buffer Eye and skin irritant. Respiratory and digestive tract irritant. Goggles, gloves. Flush eyes with water at eye-wash sink. Wash skin with water for 15 minutes. Remove contaminated clothing/shoes. Do not induce vomiting. Drink up to 4 cups of water or milk. If breathing becomes problematic oxygen should be given, otherwise move to fresh air. In case of spill absorb with inert material, wear protective gear, provide ventilation. Store in tight containers.
Iron reagent (3M Nitric Acid) Irregular heart beat

Dizziness, coughing, sore throat, vomiting. Corrosive burns to skin, eyes and respiratory tract

Goggles, gloves, labcoat Wash immediately with water. Flush eyes with water for 15 minutes. Seek professional medical assistance

Absorb spills with inert material e.g. Sand, spill kit. Place into a closed container for disposal

Sealed container. Not near combustibles.
Mercury (organic) Major irritant of skin and eyes. Toxic to blood, kidneys, liver, brain and nervous system. Mask, gloves, work in fumehood Do not breath gas or fumes. Wash skin and eyes with running water. Spills must immediately be wiped with inert material and placed in dedicated waste container May react with other substances to be explosive. Keep separated from other chemicals.


Protocol: Heat-shock Transformation of Bacterial Cells
Name Risk Required Protection Equipment First Aid Response Storage
TE Buffer Contains HCl. Can be absorbed through skin, causing reddening and/or inflammation. Irritant of the eyes and respiratory system. Goggles, gloves. Flush eyes with water at eye- wash sink. Wash skin with water for 15 minutes. If inhaled, immediately move away from site and get fresh air. If having difficulty breathing may require oxygen. Spills: use absorbent but inert material and dispose of into contaminated waste containers. Contaminated area must then be wiped down with detergent and rinsed thoroughly with water. Store in tight containers.
Ice. Cold Cells (-80°C) Ice may cause cold burns. Melted ice may cause electrical hazard Goggles, thermal gloves. Run skin under tepid water. If electrocuted turn of the electric device. Check for normal breathing. If a burn is attained treat according to the degree of the burn. Notify a supervisor immediately work with ice in waterproof container. Keep away from electrical equipment.


Protocol: Plasmid MiniPrep
Name Risk Required Protection Equipment First Aid Response Storage
TE Buffer Contains HCl. Can be absorbed through skin, causing reddening and/or inflammation. Irritant of the eyes and respiratory system. Goggles, gloves. Flush eyes with water at eye- wash sink. Wash skin with water for 15 minutes. If inhaled, immediately move away from site and get fresh air. If having difficulty breathing may require oxygen. Spills: use absorbent but inert material and dispose of into contaminated waste containers. Contaminated area must then be wiped down with detergent and rinsed thoroughly with water. Store in tight containers.
Sodium Dodecyl Sulfate (SDS) An allergen in some individuals. Causes irritation to eyes and skin. Dehydrates mucosal membrane if ingested Goggles, gloves. Flush exposed areas with water for approx. 20 minutes. If allergic immediately get emergency aid. Store away from heat or sources of ignition
NaOH Causes severe irritation to eyes and skin. Causes chemical burns to respiratory and intestinal tract Goggles, gloves. Work in Fumehood Remove contaminated clothing. Wash skin/eyes with running water for 20 minutes. If inhaled quickly move to fresh air.

Do not induce vomiting if ingested but drink plenty of water. Never dispose of material in sinks. Vacuum if spilt or gently sweep away.

Must not be stored in aluminium, zinc, tin, lead or glass containers. Do not store with acids, oxidisers and chlorinated organic compounds
Potassium Acetate (KAc) Irritant to eyes and skin. Goggles, gloves. Wash skin with non-abrasive soap. Cover skin with lotion. Flush eyes with cold water for 20 minutes and remove any contacts if being worn. Loosen tight clothing if ingested. Neutralise K-Ac with dilute acetic acid. Wipe down with water. In cool temperatures away from sources of ignition.
Phenol Contact and inhalation highly corrosive to skin and respiratory tract. Some systemic toxicity. Goggles, gloves. Work in fumehood. Wash skin with a disinfectant soap and cover with an anti-bacterial cream. Flush eyes with water for 20 min. If large splash seek urgent medical attention. Store away from light, heat and ignition sources.
Ethanol Irritant of skin and eyes. Vapors may cause nausea, headaches or dizziness. Flammable. Goggles, gloves. Work in fumehood. If ingested drink 3 cups of milk. Seek fresh air if causing nausea. Wash skin and/or eyes with water for 15 minutes. Store away from heat and ignition sources.
Chloroform: Isoamyl Causes inflammation. Vapors may cause nausea, headaches or dizziness. Flammable. Goggles, gloves. Work in fumehood. If inhaled exit the room and get fresh air. Store away from heat and ignition sources.


Protocol: Preparation of Competent Cells
Name Risk Required Protection Equipment First Aid Response Storage
Potassium Chloride (KCl) Can cause chemical conjunctivitis. Nausea, vomiting and diarrhoea from ingestion. Cause electrolytic imbalance. Goggles, gloves. Wash skin or eyes with water for 15 minutes. Remove contaminated clothing and wash before reuse. If ingested drink one cup of milk. Medical attention may be required if more serious. Store in dry and cool area as it is hygroscopic.
Manganese Chloride (MnCl2) Inhalation causes metal-fume fever. Targets reproduction and nervous systems. Irritant of skin and eyes. Causes vomiting and nausea. Cause of argyria (bluish tinge will appear on skin) Goggles, gloves. If ingested drink 2 cups milk. Wash skin and eyes with water for 15 minutes. Medical attention may be necessary. Clean up spills by vacuuming. Keep area well ventilated. Store away from incompatible substances., Store in dry and cool area
Calcium Chloride (CaCl2) May cause coughing and shortness of breath if inhaled. Reacts with mucosal membrane to cause damage or irritation to tissue lining. May experience nausea or vomiting. Goggles, gloves. Skin should be washed with WARM water. Flush warm water with tepid water. If ingested drink 1-2 glasses of water Store in dry, well ventilated area. Do not reuse bottle previously containing CaCl2.
Sodium Hydroxide (NaOH) Causes severe irritation to eyes and skin. Cause of chemical burns to respiratory and intestinal tract Goggles, gloves. Remove contaminated clothing. Wash skin/eyes with running water for 20 minutes. If inhaled quickly move to fresh air.

Do not induce vomiting if ingested but drink plenty of water. Never dispose of material in sinks. Vacuum if spilt or gently sweep away.

Must not be stored in aluminium, zinc, tin, lead or glass containers. Do not store with acids, oxidisers and chlorinated organic compounds.
Magnesium Chloride (MgCl2) Mild irritant of mucosal membranes. Absorbed slowly via skin so diarrhoea or nausea may be experienced hours later. Hypocalcaemia Goggles, gloves. Drink many glasses of water to dilute. Wash skin with mild soap. In case of spills, provide good ventilation. Pour water over the area to prevent dispersal of substance in air. Sweep and put into closed container for disposal. Tightly sealed containers to prevent distribution in air.
Magnesium Sulfate (MgSO4) Mild irritant of mucosal membranes. Absorbed slowly via skin so diarrhoea or nausea may be experienced hours later. Hypocalcaemia Goggles, gloves. Drink many glasses of water to dilute. Wash skin with mild soap. In case of spills, provide good ventilation. Pour water over the area to prevent dispersal of substance in air. Sweep and put into closed container for disposal. Tightly sealed containers to prevent distribution in air.
Protocol: SDS-PAGE
Name Risk Required Protection Equiptment First Aid Response Storage
Acrylamide Neurotoxic, Carcinogenic, Mutagenic. Goggles, gloves. Flush eyes with water. Skin needs to be washed with non-abrasive soap. Remove contaminated clothing. Spills must be wiped using inert material. Dispose of material in appropriate contaminated waste. Water must be spread over contaminated surface. Store in tight containers away from light and heat.
TEMED Toxic, Corrosive. Goggles, gloves. Flush eyes with water. If moderate to high exposure to skin made seek medical help. Remove contaminated clothing. Spills must be wiped using inert material. Dispose of material in appropriate contaminated waste. Water must be spread over contaminated surface. Store in tight containers away from light and heat.
Sodium Dodecyl Sulfate (SDS) An allergen in some individuals. Causes irritation to eyes and skin. Dehydrates mucosal membrane if ingested Goggles, gloves. Flush exposed areas with water for approx. 20 minutes. If allergic immediately get emergency aid. Store away from heat or sources of ignition


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 and any equipment it came into contact with.
  • 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 thiocyanate, 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.

With thanks to:



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