Application Title: Modification of a Burkholderia Soil Bacteria for The

NO3P Develop in containment a project of low risk genetically ER-AF-NO3P-3 modified organisms by rapid assessment 12/07

Application title:

Modification of bacteria for the creation of a bacterial biosensor to detect and quantify various compounds or elements.

Applicant organisation: Environmental Science and Research LTD.

Considered by:

IBSC ERMA √

Please clearly identify any confidential information and attach as a separate appendix.

Please complete the following before submitting your application: All sections completed Yes Appendices enclosed Yes/NA Confidential information identified and enclosed separately Yes/NA Copies of references attached Yes/NA Application signed and dated Yes Electronic copy of application e-mailed to ERMA New Yes Zealand

Signed: Joanna Lloyd Date:24/06/2009

20 Customhouse Quay Cnr Waring Taylor and Customhouse Quay PO Box 131, Wellington Phone: 04 916 2426 Fax: 04 914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Develop in containment a project of low risk genetically modified organisms by rapid assessment

1. An associated User Guide NO3P is available for this form and we strongly advise that you read this User Guide before filling out this application form. If you need guidance in completing this form please contact ERMA New Zealand or your IBSC. 2. This application form only covers the development of low-risk genetically modified organisms that meet Category A and/or B experiments as defined in the HSNO (Low-Risk Genetic Modification) Regulations 2003. 3. If you are making an application that includes not low-risk genetic modification experiments, as described in the HSNO (Low-Risk Genetic Modification) Regulations 2003, then you should complete form NO3O instead. 4. This form replaces all previous versions of Form NO3P. 5. This application form may be used to seek approvals for more than one new organism where the organisms are used in the same project, or have a similar risk profile. 6. Any supporting material that does not fit in the application form must be clearly labelled, cross- referenced, and included as appendices to the application form. 7. Commercially sensitive information must be collated in a separate appendix but referenced in the application. You need to justify why you consider the material commercially sensitive, and make sure it is clearly labelled as such. Confidentiality of material is subject to the provisions of the Official Information Act 1982 and the basis of which is that information should be publicly available unless there is good reason to protect it. 8. Applicants must sign the form and enclose the correct application fee (plus GST) if it is submitted to ERMA New Zealand. Details of the application fee can be found in our published Schedule of Fees and Charges. Please check with ERMA New Zealand staff or the ERMA New Zealand website for the latest schedule of fees. 9. Unless otherwise indicated, all sections of this form must be completed for the application to be progressed. 10. Please provide an electronic version of the completed application form, as well as sending a signed hard copy. You can get more information by contacting your Institutional Biological Safety Committee or ERMA New Zealand. This version of the application form was approved by the Chief Executive of ERMA New Zealand on 12 November 2007.

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Section One – Applicant details refer to page 9 of the user guide Name and details of the organisation making the application: Name: Environmental Science and Research LTD Postal Address: Po Box 50-348 Physical Address: 34 Kenepuru Drive Phone: 04 9140700 Ext 7814 Fax: 04 9140700 Email: Name and details of the key contact person (if different from above): Name: Joanna Lloyd Postal Address: Physical Address: Phone: Fax: Email: [email protected] Name and details of a contact person in New Zealand, if the applicant is overseas: Name: Postal Address: Physical Address: Phone: Fax: Email:

Note: The key contact person should have sufficient knowledge of the application to respond to queries from ERMA New Zealand staff.

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Section Two: Lay summary and scientific project description refer to page 9 of the user guide

Lay summary of the application (approximately 200 words)

Note: This summary should describe the genetically modified organism(s) being developed, the purpose of the application or what you want to do with the organisms(s). Use simple non- technical language.

Environmental Science and Research LTD have previously contributed to research into the application and use of bacteria for the detection and quantification of organic and inorganic compounds [1, 2]. This current research project will allow the real-time detection and quantification of various compounds/elements thereby enabling enhanced environmental protection and management. The organism will be developed using a highly specific, genetically modified (GM) non native model bacterium such as Escherichia coli. Biologically-based recognition mechanisms will be used such as a reporter gene that can visually detect when the organism encounters the compound in question.

This research will involve no native or human genetic material. Genetic modification of these developed organisms is considered “low risk” and any genetic modification performed under this application will not increase the pathogenicity of these organisms or increase their ability to form a damaging self sustaining population. The modified organisms are limited to the approved work area by stringent laboratory protocols already in place.

Scientific project description (describe the project, including the background, aims and a description of the wider project) refer to page 10 of the user guide

Note: This section is intended to put the genetically modified organism(s) being developed in perspective of the wider project(s) that they will be used in. You may use more technical language but make sure that any technical words are included in the Glossary.

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Objective:

To develop in containment a bacterial biosensor (a bacteria with the ability to provide a measurable response to a particular compound(s) or element(s).

Proposed research: Bacterial biosensors are biological tools that can probe the environment for the presence of compounds. They have been compared to the traditional „canaries in a mine‟, as they can detect chemicals rapidly, safely, and at very low concentrations. For example, studies have shown that the organic compound, 1080, can be metabolised by soil microorganisms, such as Pseudomonas and Fusarium species [3] . The gene sequences for the enzymes capable of defluorinating 1080 have been isolated [4]. We will couple defluorination genes with a reporter gene (e.g. bacterial bioluminescence or Green Fluorescent Protein) creating a genetically modified bacteria that „turns on‟ reporter gene expression in response to exposure to a particular compound or element , to give an optically detectable signal.

Naphthalene degradation and heavy metal resistance genes have previously been isolated, linked to lux-reporter genes and developed into specific biosensors, thus the technology is known and can be applied to the construction of a specific biosensor [5]. The sensitivity of these kinds of sensors is at sub-toxic concentrations, thus, the working range (and thus the sensitivity) is remarkably low. In the case of a specific biosensor for the heavy metal cadmium, the limit of detection was found to be 0.0008 mg/l, we would expect the biosensor to have similar sensitivity [6].

This research programme will enhance environmental management of various compounds or elements, e.g. iron. To achieve this, we will develop a highly specific, bacterial biosensor that emits light in direct proportion to the concentration of the compound or element present. The system will complement conventional chemical analysis by providing a rapid (< 60 min), inexpensive (< $100) preliminary screen for various compounds or elements. In addition, the biosensor analysis will require little or no sample preparation, and have the potential for both laboratory and on-site analysis.

GMOs to be developed:

Burkholderia and/or E. Coli and/or Actinobacter and/or Campylobacter and/or Pseudomonas bacteria will be modified with a standard commercial plasmid vector or a specifically designed plasmid vector containing bacterial DNA (derived from either risk group 1 or risk group 2 bacteria) for example a DNA sequence (promoter) isolated from Burkholderia spFA1 which is capable of quantitatively responding to 1080 concentrations. This DNA sequence will be fused to reporter genes such as a “light producing” gene (lux) or a Green Fluorescent Protein (GFP) gene. Non native Bacterial DNA (risk group 1 or 2) will be sourced from culture collections or from environmental samples (with the necessary approval if required).

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Short summary of purpose (please provide a short summary of the purpose of the application) (255 characters or less, including spaces) refer to page 11 of the user guide. This section will be transferred into the decision document.

To develop in containment genetically modified bacteria that can detect and quantify compounds and/or elements.

Section Three –Description of the organism(s) to be developed refer to page 13 of the user guide

3.1 Identification of the host organism to be modified

Complete this section separately for each host organism to be modified.

Latin binomial, including full Burkholderia cepacia (Palleroni and Holmes taxonomic authority: 1981) Yabuuchi et al. 1993

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

Strain(s) if relevant: Strain: Burkholderia cepacia complex genomovar I. NCTC 10743 ATCC 25416 DSM 7288 CDC KC984

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

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Latin binomial, including full Burkholderia stabilis taxonomic authority: Vandamme et al 2000

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

Strain(s) if relevant: Strain: 4015T LMG 10929 ATCC: BAA-67 NCTC 13011 Burkholderia cepacia complex genomvar IV.

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Burkholderia vietnamensis taxonomic authority: Gillis et al 1995

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

Strain(s) if relevant: Strain: 4016T Burkholderia cepacia complex genomvar V. LMG 10929

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DSM 11319

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Escherichia coli taxonomic authority: (Migula, 1895; Castellani and Chalmers, 1919).

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Eubacteria, Protobacteria, Gammaproteobacteria, Enterobacterials, Enterobacteriaceae

Strain(s) if relevant: Non-pathogenic laboratory strains of Escherichia coli, in particular K-12 (DH5alpha Lambda PIR fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17).

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Campylobacter coli (Doyle, 1948) Veron and taxonomic authority: Chatelain, 1973

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria, Epsilonproteobacteria Campylobacterales, Campylobacteraceae Campylobacter

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Strain(s) if relevant: Strain 2607T NCTC 11366 ATCC: 33559 DSM 4689 CIP 7080

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Burkholderia glathei taxonomic authority: (Zolg and Ottow 1975) Vandamme et al. 1997

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

Strain(s) if relevant: 50012, 50014T

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Burkholderia spp taxonomic authority: (Zolg and Ottow 1975) Vandamme et al. 1997

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

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Taxonomic class, order and family: Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

6431, 6613, 6756, 6818, 7116, 7336, 8530, 9242, 9243, 13785, 15090, Strain(s) if relevant: 19529, 50401

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Campylobacter fetus (Smith & Taylor 1919) taxonomic authority: Sebald & Véron 1963

Common name(s), if any:

Type of organism (eg bacterium, Microorganism / bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria, Epsilonproteobacteria Campylobacterales, Campylobacteraceae Campylobacter

Strain(s) if relevant: Strain: 5 ATCC: 10702 DSM 487 NCIB 8122

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Pseudomonas fluorescens taxonomic authority: Migula 1895

Common name(s), if any:

Type of organism (eg bacterium, Gram negative, non-spore forming bacterium virus, fungus, plant, animal, animal cell):

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Taxonomic class, order and family: Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas

Strain(s) if relevant: Strain 862T ATCC: 13525, DSM 50090, NCIB 9046, NCTC 10038

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Pseudomonas putida taxonomic authority: (Trevisan 1889) Migula 1895

Common name(s), if any:

Type of organism (eg bacterium, Gram negative, non-spore forming bacterium virus, fungus, plant, animal, animal cell):

Taxonomic class, order and family: Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas

Strain(s) if relevant: Strain 852 ATCC 12633 DSM 291 NCIB 9494 NCTC 10936

Other information, including No inseparable or associated organisms present. presence of any inseparable or associated organisms and any related animals present in New Zealand:

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3.2 Information on the host organism

Refer to pages 14-19 and pages 33-38 of the user guide for assistance in completing this section Complete this section separately for each host organism to be modified.

Burkholderia Strains Yes No

1 Is the organism normally capable of causing disease in humans, Yes animals, plants or fungi? Organisms from the Burkholderia complex are important pathogens in cystic fibrosis and have been isolated in the sputum of patients with Cystic Fibrosis lung disease, however this bacteria is not normally a pathogen of health individuals [7]. B cepacia is also a known cause of infections in hospitalized patients. B. stablis strains have also been isolated from CF patients [7]. In these cases, multiple antibiotics are available for treatment. The genetic modifications will not result in resistance to antibiotics used for the treatment of infections caused by Burkholderia strains.

2 Is the organism a human cell line? No If yes, provide details here of where the material has been obtained from and whether approval has been obtained from an Ethics Committee (if required) 3 Is the organism native to New Zealand? No If yes, provide details here for example, from where will this material be obtained? Be as specific as possible as this information may be needed to determine whether Māori have been consulted appropriately 4 Does the organism contain infectious agents normally able to cause No disease in humans, animals, plants or fungi? If yes, provide details here.

5 Does the organism produce desiccation resistant structures (such as No spores or cysts) that can normally be disseminated in the air? If yes, provide details here.

6 Is the organism characterised to the extent that its main biological Yes characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? Yes

If yes, provide details here. It may infect or colonise CF patients. Refer to answer for question 1.

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8 If the organism is a whole plant or plant tissue, do you intend to: NA a) Allow it to develop reproductive structures If yes, please provide further information on containment in section 4 b) Keep it in a closed container? 9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk No Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk Yes Genetic Modification) Regulations 2003)?

Pseudomonas fluorescens Yes No

1 Is the organism normally capable of causing disease in humans, Yes animals, plants or fungi? Members of the genus Pseudomonas are common inhabitants of soil, fresh water, and marine environments. Pseudomonas fluorescens is a plant pathogen and previously approved by ERMA New Zealand.

5 Does the organism produce desiccation resistant structures (such as No spores or cysts) that can normally be disseminated in the air? If yes, provide details here.

6 Is the organism characterised to the extent that its main biological Yes characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? No

If yes, provide details here. P. fluorescens is known to cause disease to some phytophagous nematodes eg, New Zealand grass grub. P. fluorescens can also infect hospital patients with compromised immune systems.

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Pseudomonas putida Yes No

1 Is the organism normally capable of causing disease in humans, No animals, plants or fungi?

5 Does the organism produce desiccation resistant structures (such as No spores or cysts) that can normally be disseminated in the air? If yes, provide details here.

6 Is the organism characterised to the extent that its main biological Yes characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? No

If yes, provide details here

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9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk Yes Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk No Genetic Modification) Regulations 2003)?

Campylobacter strains Yes No

1 Is the organism normally capable of causing disease in humans, Yes animals, plants or fungi? Some species of Campylobacter are pathogenic in humans and animals, and can be found in the reproductive organs, intestinal tract and oral cavity of animals and humans.

5 Does the organism produce desiccation resistant structures (such as No spores or cysts) that can normally be disseminated in the air? If yes, provide details here.

6 Is the organism characterised to the extent that its main biological Yes characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? Yes

If yes, provide details here. The common routes of transmission for Campylobacter strains are fecal-oral, person-to-person sexual contact, ingestion of contaminated food or water. Considering no food consumption is allowed in the lab and the lab protocols, the risk of human infection or colonization is low. 8 If the organism is a whole plant or plant tissue, do you intend to: NA a) Allow it to develop reproductive structures If yes, please provide further information on containment in section 4 b) Keep it in a closed container?

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9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk No Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk Yes Genetic Modification) Regulations 2003)?

Escherichia Coli Yes No

1 Is the organism normally capable of causing disease in humans, No animals, plants or fungi? E. coli K-12 and its derivatives are widely used under PC1 and PC2 containment conditions or their equivalent around the world. Its usefulness as a host for DNA cloning, amplification and expression purposes has made it one of the most widely used bacteria in Microbiology and other molecular biosciences. The risk posed to the health of the laboratory user or the wider community by E. coli K-12 is extremely low.

5 Does the organism produce desiccation resistant structures (such as No spores or cysts) that can normally be disseminated in the air? If yes, provide details here.

6 Is the organism characterised to the extent that its main biological Yes characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? No

If yes, provide details here.

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Note: If the genetic modification does not involve a Category 1 or 2 host organism then the proposed project does not meet the criteria in section 42A(2)(a) of the HSNO Act for the rapid assessment of projects for low-risk genetic modification.

3.3 Nature and range of the proposed genetic modification(s)

Refer to pages 15-19 and pages 33-38 of the user guide for assistance in completing this section Provide details on the following Complete this section separately for each host organism to be modified only if there are significant differences in the modifications for each of the host organisms listed above. Information on how the new organism(s) will be developed

Vector system used, eg cloning or Standard Commercial Plasmid vectors or expression, plasmid, or viral specifically designed plasmid vectors such as: pUCD615 as described in the reference [8], pUCD607 as described in the reference [9] , Mini Tn5-Lux CDABE as described in the reference [10]. pCS26 as described in the reference [11]. pVITIR as described in the reference [12] The vector backbone shall only contain any or all of the following elements: -Promoter, operator, regulatory element binding and enhancer sequences: -Selectable marker genes that confer the ability to: Be resistant to selected antibiotics, including kanamycin and/or tetracycline and/or ampicillin and/or spectomycin. Origins of replication: OR 6 (min Tn5), OR I (PUCD607), Ori Sa (PUCD615) and/or other pUC origins of replication.

Range of elements that the vectors The plasmid vectors pUCD615, pUCD607, Mini may contain Tn5-Lux and pCS26 already contain the Mini Tn5 Lux CDABE light producing gene cassette.

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This is used to produce luciferase enzymes and associated proteins capable of producing bioluminescence (light) as a visual reporter, additionally the vectors contain Antibiotic resistance genes (used for screening/selection of the bacteria). The plasmid vector pVITIR contains a Green Fluorescent Protein gene cassette, which confers the ability to produce Green Fluorescent Protein which is detectable with U.V light

Type, source and function of any Non native Bacterial DNA involved in the donor genetic material recognition, of various compounds or elements for example, sodium monofluoracetate and iron. For example, in more detail, PCR products of the promoter and regulatory element binding sequences for a Dehalogenase gene derived from genomic DNA of a Burkholderia sp FA1 as described in the reference [4]. Non native bacterial DNA regulated by the presence and absence of iron. The DNA will be inserted into (if not already present) any or all of the plasmids mentioned above for characterisation in one of the aforementioned bacteria. All donor DNA will have undergone bioinformatic assessment to ensure the candidate genes are well characterised and that high risk DNA elements are not being introduced.

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Use of special genetic material

Yes No

Does the proposed modification use genetic material derived from no organisms capable of causing disease in humans, animals, plants or fungi?

If yes, provide details here including the sequences as well as the species and strains they were derived from. If the genetic material to be introduced is characterised so that its sequence and gene function are known, please state this

Does the proposed modification use genetic material from native biota? no

If yes, provide details here including where this material will be obtained from. Be as specific as possible as this information may be needed to determine whether Māori have been consulted appropriately

Does the proposed modification involve human genetic material? Answer no yes if human genetic material in any form is used, ie whether it is obtained directly from humans, from a gene bank, synthesised, copied and so on.

Other details of the modification, including any unusual manipulations, if the foreign genetic material is to be expressed, where it is expected to be expressed and what techniques will be used in the modification. The bacteria will be modified by: Transformation of plasmids for extrachromosomal replication and the specific promoter induced expression of reporter genes, or the transformation of plasmids which confer the ability to insert into the chromosome of the host for the specific promoter induced genetic expression of reporter genes. Non native Bacterial DNA is sourced from overseas or from our bacterial culture collection at ESR or alternatively from environmental samples such as soil.

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3.4 Identify the category of experiments as described in the HSNO (Low-Risk Genetic Modification) Regulations, 2003. Refer to pages 17-19 and pages 33-38 of the user guide for assistance in completing this section.

Burkholderia Species Yes No

Is the proposed modification to a Category 1 host organism? No 1

Is the proposed modification to a Category 2 host organism? Yes 2

Will the proposed modification increase the pathogenicity, virulence, No 3 or infectivity of the host organism to laboratory personnel, the community, or the environment? If you answer yes to this question, please confirm with an ERMA advisor that the modification is low risk.

Will the proposed modification result in a genetically modified No 4 organism with a greater ability to escape from containment than the unmodified host? If you answer yes to this questions, please confirm with an ERMA advisor that the modification is low risk.

Is the proposed modification to be carried out under a minimum of No 5 PC1 containment?

Is the proposed modification to be carried out under a minimum of yes 6 PC2 containment?

Does the proposed modification conform to the requirements of a No 7 Category A genetic modification?

Does the proposed modification conform to the requirements of a Yes 8 Category B genetic modification?

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Pseudomonas fluorescens Yes No

Is the proposed modification to a Category 1 host organism? No

Is the proposed modification to a Category 2 host organism? Yes

Will the proposed modification increase the pathogenicity, virulence, or No infectivity of the host organism to laboratory personnel, the community, or the environment? If you answer yes to this question, please confirm with an ERMA advisor that the modification is low risk.

Will the proposed modification result in a genetically modified organism No with a greater ability to escape from containment than the unmodified host? If you answer yes to this questions, please confirm with an ERMA advisor that the modification is low risk.

Is the proposed modification to be carried out under a minimum of PC1 No containment?

Is the proposed modification to be carried out under a minimum of PC2 yes containment?

Does the proposed modification conform to the requirements of a Category No A genetic modification?

Does the proposed modification conform to the requirements of a Category Yes B genetic modification?

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Campylobacter species Yes No

Is the proposed modification to a Category 1 host organism? No

Is the proposed modification to a Category 2 host organism? Yes

Will the proposed modification increase the pathogenicity, virulence, No or infectivity of the host organism to laboratory personnel, the community, or the environment? If you answer yes to this question, please confirm with an ERMA advisor that the modification is low risk.

Will the proposed modification result in a genetically modified No organism with a greater ability to escape from containment than the unmodified host? If you answer yes to this questions, please confirm with an ERMA advisor that the modification is low risk.

Is the proposed modification to be carried out under a minimum of No PC1 containment?

Is the proposed modification to be carried out under a minimum of yes PC2 containment?

Does the proposed modification conform to the requirements of a No Category A genetic modification?

Does the proposed modification conform to the requirements of a Yes Category B genetic modification?

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E. coli and Pseudomonas putida Yes No

Is the proposed modification to a Category 1 host organism? Yes

Is the proposed modification to a Category 2 host organism? No

Is the proposed modification to be carried out under a minimum of No PC1 containment?

Is the proposed modification to be carried out under a minimum of yes PC2 containment?

Does the proposed modification conform to the requirements of a yes Category A genetic modification?

Does the proposed modification conform to the requirements of a No Category B genetic modification?

Explanation of categorisation, if necessary. This is particularly important for work involving pathogenic microorganisms and viral vectors

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Section Four – The proposed containment system Refer to page 20 of the user guide for assistance in completing this section Describe the containment facility and the proposed containment system (physical and operational) Question Answer Which MAF/ERMA Standard is this Standard 154.02.17 and 154.03.02 containment facility approved under? Facilities for Microorganisms and Cell Cultures 2007a What physical containment level Physical Containment Level 2 (PC 2) as defined by (AS/NZS 2243.3:2002) is this The Australian/New Zealand Standards (AS/NZS containment facility approved to 2243.3:1995). These correspond to a low likelihood operate at (where relevant)? of escape.

What other physical measures do you A Class II cabinet, used for bacterial propose to use to contain this manipulations, is also attached to an uninterrupted organism? power supply to prevent escape of any organisms by aerosol in the advent of a power outage during experimental procedures with bacterial cultures.

What procedural or operational All personnel handling GMO‟s will receive measures do you propose to use to training and wear appropriate safety clothing such contain this organism? as gloves and lab coats. Such small amounts of organism being used in individual experiments (maximum of 1L at a cell density of 10-9 cells ml-1) means that there would be no predicted environmental perturbation as the numbers already in the environment are much greater. The GMO require special growth conditions to survive in the environment. They can also be destroyed using 10% bleach or by autoclaving. The genetic modification of the organism could not be expected to increase the ability of the organism to colonise any particular environmental habitat or niche. To eliminate the risk of escape of bacterial samples, sterilisation of all wastes is carried out by autoclave prior to disposal

Any other information relevant to the Long-term storage of the modified organisms will containment of the organism. be in glycerol stocks in 1.5ml tubes in -20 degree freezers in PC2 containment at ESR Transitional Facilities for Microorganisms and Cell cultures in Kenepuru.

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Section Five – Identification and assessment of adverse effects Refer to page 21 of the user guide for assistance in completing this section This section should only be completed in detail if pathogenic microorganisms, human cells, native or valued flora and fauna were identified as host or sources of donor genetic material in section 3. It is expected that organisms meeting the low-risk regulations will not normally have any significant biological risks associated with them. However, there may still be some adverse effects that need to be identified and assessed. This might include economic, social and cultural adverse effects and other risks not addressed by the HSNO (Low-Risk Genetic Modification Regulations) 2003 What adverse effects could this organism have on the environment? For all stages of the life cycle The containment facilities to be used at ESR are secure and precautions (as described in section Four) are in place to prevent possible escape therefore no significant adverse effects have been identified.

What adverse effects could this organism have on human health and safety?

No significant adverse effects have been identified in normal healthy individual‟s, however Burkholderia cepacia (although not normally a pathogen) may colonize and/or infect the respiratory tract of patients with cystic fibrosis [7] and Pseudomonas fluorescens can infect hospital patients with compromised immune systems as stated in ERMA approval GMD08071 (we note that infection was via contaminated saline and it is highly improbably under the MAF/ERMA New Zealand standards that infection will occur). Research will be conducted in a PC2 facility with the appropriate protective clothing and equipment such as wearing a laboratory coat and gloves. The AS/NZS 2243:2002 recognizes Burkholderia cepacia, Campylobacter coli, and Campylobacter fetus as risk group 2 bacteria (Table 3.1) and does not require any special precautions. A Class II cabinet, used for bacterial manipulations, is also attached to an uninterrupted power supply to prevent escape of any organisms by aerosol in the advent of a power outage during experimental procedures with bacterial cultures. Taking into account the staff PC2 training and the appropriate protective clothing and equipment, the likelihood of infection is low.

E. coli is very widely used as a host for DNA from microorganisms isolated from the environment without causing concerns with regard to human health and safety therefore no significant risks to human health have been identified with this organism for this type of investigation.

Genetic modification of these organisms is considered “low risk” and any genetic modification performed under this application will not increase the pathogenicity of these organisms. The modified organisms are limited to the approved work area by stringent laboratory protocols already in place to prevent contamination of areas outside PC2 containment facility. The inserted DNA represents a sequence of DNA that is able to recognise various compounds or elements.

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All waste material is sterilised by high pressure heating (autoclaving) with disposal according to infectious waste disposal protocols already in-place at ESR.

What adverse economic effects could this organism have?

No significant adverse effects have been identified.

What adverse effects could this organism have on the relationship of Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga (taking into account the principles of the Treaty of Waitangi)? According to the “Working with Maori under the HSNO Act 1996” guide, under the general criteria listed in Table 1 their are no environmental or economic development and well-being outcomes of significance to iwi/Māori since this work will be in conducted in containment. The development of the GMOs do not involve any native or human genetic material so there are not cultural outcomes or health and well being outcomes of significance to iwi/Māori. Therefore there are no potential adverse effects on the relationship of Maori and their culture and traditions with their ancestral lands, water, sites waahi tapu, valued flora and fauna and other taonga were identified for this application.

Are there any other potential adverse effects? No potential adverse effects have been identified.

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Section Six – Additional information Refer to page 31 of the user guide for assistance in completing this section Additional Information Y/N If yes, explain Do any of the organism(s) need N approvals under any other New Zealand legislation? Does New Zealand have any N international obligations relating to (any of) the organism(s)? Have any of the new organism(s) in Y GMD02106 – E.coli for monitoring this application previously been environmental samples considered in New Zealand or GMC99004 – to import bacteria elsewhere? Is there any additional information N that you consider relevant to this application that has not already been included? Following the development of this A lab based tool for the detection and organism what will the genetically quantification of various compounds or modified organism be used for? eg elements in a variety of mediums, will experimental animals or plants including soil, water and sediments in order be exposed to this organism? to investigate the environmental fate of various compounds or elements in containment.

Provide a glossary of scientific and technical terms used in the application:

 Biosensors: a bacteria with the ability to provide a measurable response to a particular substance.  Plasmid: an extra chromosomal ring of DNA especially of bacteria that replicates autonomously.  DNA: nucleic acids that are usually the molecular basis of heredity, are localised especially in cell nuclei, and are constructed of a double helix held together by hydrogen bonds between purine and pyrimidine bases which project inward from two chains containing alternate links of deoxyribose and phosphate.  Expression: the detectable effect of a gene.  PCR: Polymerase chain reaction, a method for amplifying DNA segments.  Gene: a specific sequence of nucleotides in DNA or RNA that is located usually on a chromosome and that is the functional unit of inheritance controlling the transmission and expression of one or more traits by specifying the structure of a particular polypeptide and especially a protein or controlling the function of other genetic material.

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List of references attached:

1. Redshaw, N., et al., A preliminary investigation into the use of biosensors to screen stomach contents for selected poisons and drugs. Forensic Science International, 2007. 172(2-3): p. 106-111. 2. Horswell .J, D.S.J., Use of Biosensors to Screen Urine Samples for Potentially Toxic Chemicals. Journal of Analytical Toxicology, 2003. 27: p. 372-376. 3. Wong D.H, et al., Defluorination of sodium monofluoroacetate (1080) by microorganisms isolated from western Australian soils. Soil Biology and Biochemistry, 1992. Volume 24(9): p. 833-838. 4. Kurihara, T., et al., Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1. Journal of Molecular Catalysis B: Enzymatic, 2003. 23(2-6): p. 347-355. 5. Heitzer, A., et al., Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. Applied and Environmental Microbiolgy., 1994. 60: p. 1487–1494. . 6. Tauriainen, S., et al., Luminescent bacterial sensor for cadmium and lead. Biosensors and Bioelectronics, 1998. 13: p. 931–938. 7. Burke, A.C. Burkholderia. Chief Infectious Disease Divison Winthrop-University Hospital 2008 [cited 4th June 2009]; Available from: http://emedicine.medscane.com/article/237122. 8. Rogowsky, P.M., et al., Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol, 1987. 169(11): p. 5101-12. 9. Rattray, E.A.S., et al., Luminescence-Based Nonextractive Technique for In Situ Detection of Escherichia coli in Soil. Applied and Environmental Microbiology, 1990. 56(11): p. 3368-3374. 10. Hedda Weitz .J, et al., Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing. FEMS Microbiology Letters, 2001. 197(2): p. 159-165. 11. Beeston, A.L. and M.G. Surette, pfs-dependent regulation of autoinducer 2 production in Salmonella enterica serovar Typhimurium. J Bacteriol, 2002. 184(13): p. 3450-6. 12. Joyner, D.C. and S.E. Lindow, Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor. Microbiology, 2000. 146 ( Pt 10): p. 2435-45.

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