Transformation of E.coli with pFluoroGreen

Transformation of E.coli with pFluoroGreen: What risks are to staff setting this prac up?  We are only a basic PC1 school.   Could you please send us a reply with information regarding any concerns? 

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Publication Date: 07 June 2016
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Transformation of E.coli with pFluoroGreen

In brief:

This activity has a number of different safety and legal aspects to consider prior to purchasing and conducting.

  • Legal requirements: This activity demonstrates a procedure known as genetic transformation. All genetic modifications are regulated by the Office of the Gene Technology Regulator (OGTR) to ensure that the required facilities are used and procedures are followed1. Whilst this activity may be considered an exempt dealing, there are still requirements for facilities to be the equivalent of a PC1 laboratory and the genetically modified organisms (GMOs) not to be released into the environment2.
  • Staff training: staff should be highly trained in good microbiological laboratory practice and have a good understanding of the gene technology involved in this activity. They should have a good understanding of responsible and safe handling of microorganisms, able to recognize biological hazards, recognize if a culture has become contaminated and give a high level of supervision to student activities to ensure correct procedures are implemented. Non- specialist teachers should not carry out this work.
  • Subculturing: This activity requires subculturing in the form of transferring E.coli from tubes and plates by streaking and pipetting. Subculturing is a specialised technique requiring sound knowledge and expertise to minimise the risks involved. It is a skill developed with much practice. Many jurisdictions do not allow microorganisms to be subcultured. Once students have prepared a culture it is not recommended that these plates are opened for any manipulation due to the risk of contamination and growing unknown microorganisms.
  • Incubation temperature: The recommended temperature for the incubation of microorganisms in schools is at room temperature or up to a maximum of 30°C and NOT 37°C to minimise the likelihood for growth of potential human pathogens that are adapted to human body temperature.3 This activity requires growth at 37oC as the E.coli will not grow well at temperatures below this.
  • Choice of microorganism: this should be a strain of E. coli, which is classified as a Risk Group 1 micro-organism which is unlikely to harm human health in healthy individuals. However this may be a risk to people who are immunocompromised or immunosuppressed. Ensure that the host bacteria E.coli is a RG1 micro-organism which is a non-pathogenic strain.
  • Selective media: The type of media used in schools should not be selective or enriched agars which may encourage the growth of pathogens3. Nutrient agar is a simple media which supports the growth of a wide variety of bacteria and moulds and is suitable for use in school laboratories. In this activity the addition of ampicillin to the agar generates a selective medium which is required to allow the bacteria containing the gene for ampicillin resistance to grow. Any additional chemicals added to the agar should have a risk assessment conducted regarding their suitability for use in schools.
  • Penicillin allergies: Ampicillin which is added to the agar is a member of the penicillin family of antibiotics. Staff and students who may be allergic to penicillin should avoid all contact.
  • Sterilisation and decontamination: The antibiotic resistant, genetically modified bacteria produced need to be destroyed and not released into the environment. The method suggests using 10% bleach as an alternative if an autoclave is not available. Science ASSIST recommends the use of a pressure cooker or autoclave for sterilising rather than chemical sterilisation, which has risks and limitations. For information regarding sterilising agar see AIS: Sterilising Agar.

Additional information:

Licencing Requirements: In Australia the Gene Technology Act 2000 and the Gene Technology Regulations 2001, along with state laws have been developed to protect the health and safety of people and the environment through regulating certain dealings and activities with genetically modified organisms (GMOs).

The Australian Government has within the Department of Health established the Office of the Gene Technology Regulator (OGTR) which has legislative power to enforce requirements under the Gene Technology Act 2000 where work involving certain dealings with gene technology is being undertaken. All dealings with genetically modified organisms (GMOs) must be licensed, notified or exempt by law.

Updated: 28 June 2018

For information on activities regarding GMOs in schools, see the "GM kits in Schools" fact sheet available on the following webpage: http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/factsheets

This information replaced an existing document titled "Regulation of gene technology in Australian schools" that was removed in June 2018. The document can still be viewed through the Internet Archive's Wayback Machine at the URL: https://web.archive.org/web/20180318031422/http://www.ogtr.gov.au/intern...$FILE/fact-gmos-in-schools.pdf This fact sheet has further links to more information including guidance notes for the containment of exempt dealings, which specifies the required facilities and procedures required. The viability of these links cannot be guaranteed by Science ASSIST.

End Update

Science ASSIST recommends that before schools embark on working with microorganisms they should ask the following questions and perform a site specific biological risk assessment:

  • Do the school facilities comply with the requirements of PC1 laboratories? Generally, Australian school science laboratories are classified as Physical Containment level 1 (PC1) and this is only if they conform to the requirements specified in Section 5 of AS/NZS 2243.3:2010 Safety in Laboratories – Microbiological safety and containment. At this level they are only suitable for work with microorganisms where the hazard levels are low, and where laboratory personnel can be adequately protected by standard laboratory practice3. Microorganisms that are classified as Risk Group 1 are the only group that should be handled in PC1 laboratories.
  • Does the school have the necessary equipment for sterilisation and decontamination procedures?
  • Do the staff have training in microbiological skills?
  • What microorganism is being used? Is the strain of microorganism likely to harm human health?
  • What manipulations are being performed with the microorganism? Are methods being used to eliminate or minimize exposure to potentially infectious material via aerosols, splashes, ingestion, absorption and accidental inoculation?
  • Are any staff or students wishing to participate in microbiological activities immunocompromised or immunosuppressed (Include those who are pregnant or may become pregnant, or are living with or caring for an immunocompromised individual)? These individuals are more prone to infections. If so, it has been suggested that they should consult a doctor to determine whether their participation is appropriate4.

References:

1 Regulation of Gene Technology in Australian Schools, Fact Sheet, January 2014. Australian Government, Department of Health, Office of the Gene Technology Regulator. http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/factsheets (Updated June 2018)

2 'What Dealings with GMOs are Classified as Exempt Dealings?' Australian Government, Department of Health, Office of the Gene Technology Regulator. http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/regsamend2011-3/$FILE/exemptdealings-1sept2011.pdf (September 2011)

3Society for General Microbiology. 2006. Basic Practical Microbiology: A Manual, Microbiology Online website, http://www.microbiologyonline.org.uk/file/ca2189fba3b39d24c5a44c1285d008...

4 American Society for microbiology. 2012. Guidelines for Biosafety in Teaching Laboratories, Universitat Autònoma de Barcelona website, http://www.uab.cat/doc/teaching_lab_ASM

Bio-Rad Biotechnology ExplorerTM pGLO Bacterial Transformation KitTM – Instructor’s Guide, Student Manual and Appendices http://www.bio-rad.com/webroot/web/pdf/lse/literature/Bulletin_1660033EDU.pdf

Edvotek. Transformation of E. coli with Green and Blue Fluorescent Protein Plasmids. Edvotek website. http://www.edvotek.com/222.100121.pdf (2003)

‘Microbiology’, University of Sydney WHS website, http://sydney.edu.au/whs/guidelines/biosafety/microbiol.shtml (Accessed June 2016)

Standards Australia. AS/NZS 2243.3-2010. Safety in Laboratories. Microbiology safety and containment. 2010. Sydney Australia.

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