Application to Develop Low Risk Gmos

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

Application title: Identification and characterization of Potential Methane Mitigation Technologies

Applicant organisation: AgResearch 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: Date: 9 May 2011

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: AgResearch Ltd Postal Address: Ruakura Research Centre, East Street, Private Bag 3123, Hamilton 3240 Physical Address: Phone: 07 856 2836 Fax: 07 838 5012 Email: Name and details of the key contact person (if different from above): Name: Dr Karen Wilson Postal Address: Physical Address: AgResearch (Grasslands), Palmerston North Phone: 06 351 8330 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.

Methane production from livestock is a major source of greenhouse gas emissions in New Zealand, and methane itself is more than twenty times more potent as a greenhouse gas than carbon dioxide. Reducing ruminant methane emissions is an important objective for ensuring the sustainability of ruminant-based agriculture.

The purpose of the application is to modify non-pathogenic bacteria found in rumen guts (methanogens and acetogens) and non-pathogenic bacterial hosts (eg, E. coli or B. subtilis or B. megaterium) with genes from the rumen gut bacteria to determine the function of these genes.

Methanogens are microorganisms that produce methane as a metabolic by-product in conditions where there is no oxygen thus maintaining low hydrogen levels in the rumen. Acetogens use alternative metabolic pathways that utilise hydrogen to create energy without producing methane as a greenhouse gas by-product. These microorganisms are commonly found in wetlands and in the guts of animals (eg, ruminants) and humans.

The identification of conserved and specific genes and gene features may aid in the development of suitable targets to reduce methane production in the future.

This research will involve no human or native 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.

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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The purpose of the application is to modify rumen methanogens, rumen acetogens E. coli, B.subtilis or B. megaterium hosts with rumen methanogen or rumen acetogen genes so that the function of these genes can be elucidated and thus candidate targets for methane mitigation can be identified and characterized.

Rumen methanogens principally use H2 to reduce CO2 to CH4 in a series of reactions that are coupled to ATP synthesis. The rumen harbours a variety of different methanogen species. Methanogens belong to the domain Archaea (Leahy et al, 2010).

Acetogens, in contrast, utilise H2 to produce energy with acetate as the main endproduct. While acetogens cannot compete against methanogens in a fully developed rumen, their alternative biochemical pathways (no greenhouse gas production; produce acetate which may be utilised by the ruminant, increasing animal efficiency) make them good candidates for alternative hydrogen utilizers in a methanogen free rumen.

We aim to modify Risk group 11 methanogen and acetogen species isolated from New Zealand ruminants with donor genetic material from rumen micro-organisms using standard molecular biology techniques such as PCR and DNA cloning. The ruminant micro-organism genetic material will include coding, non-coding or regulatory regions of genes involved in the methanogenesis and acetogenesis pathways. Furthermore, such genetic elements as described above will be introduced into already used heterologous microbial systems such as non-pathogenic E. coli/B.subtilis/B. megaterium strains.

Genes suitable as targets will be identified using a combination of metabolic profiling, review of the literature pertaining to the biochemistry and physiology of methanogens, and comparative genomics. Selected genetic loci will be subjected to a range of in vitro and in vivo assays to determine their structure, mode-of-action, specificity, and, ultimately, their suitability as methane mitigation targets. The developed GMOs will be characterised using a variety of common scientific techniques eg, standard staining methodology, protein expression, and microscopy.

The modifications will not include genetic material: from New Zealand indigenous fauna and flora; that increases the pathogenicity, virulence, or infectivity of the host organism; and that results in the modified organism having a greater ability to escape from containment than the unmodified host.

1 Risk Group 1: Unlikely to cause disease in humans, animals, plants and fungi.

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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 genetically modified microorganisms in containment in order to identify methanogen or acetogen specific features that would be suitable as targets for methane mitigation technologies.

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 Escherichia coli (Migula 1895) Castellani and taxonomic authority: Chalmers 1919

Common name(s), if any: E. coli

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

Taxonomic class, order and family: Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae

Strain(s) if relevant: Non pathogenic laboratory strains

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 Methanocorpusculum Zellner et al. 1988 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanomicrobia

Order: Methanomicrobiales

Family: Methanocorpusculaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Methanobrevibacter Balch and Wolfe 1981 taxonomic authority: Methanobacterium Kluyver and van Niel 1936 Methanobacter Wasserfallen et al. 2000 Methanoculleus Maestrojun et al. 1990

Methanofollis Zellner et al. 1999

Methanogenium Romesser et al. 1981

Methanolacinia Zellner et al. 1990

Methanosphaera (Miller and Wolin 1985) Type of organism Microorganism

Taxonomic class, order and family: Class: Methanobacteria

Order: Methanobacteriales

Family: Methanobacteriaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

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Latin binomial, including full Methanohalobium Zhilina and Zavarzin 1988 taxonomic authority: Methanococcoides Sowers and Ferry 1985

Methanohalophilus Paterek and Smith 1988

Methanolobus Konig and Stetter 1983

Methanomethylovorans Lomans et al. 2004

Methanomicrobium Balch and Wolfe 1981

Methanimicrococcus corrig. Sprenger et al. 2000

Methanoplanus Wildgruber et al. 1984

Methanosaeta Patel and Sprott 1990

Methanosalsum Boone and Baker 2002

Methanosarcina Kluyver and van Niel 1936 Type of organism Microorganism

Taxonomic class, order and family: Class: Methanomicrobia

Order: Methanosarcinales

Family: Methanosarcinaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

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Latin binomial, including full Methanocalculus Ollivier et al. 1998 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanomicrobia

Order: Methanomicrobiales

Family: incertae sedis

Strain(s) if relevant: Non pathogenic laboratory strains

Other information, including presence of any inseparable or associated organisms and any related animals present in New Zealand:

Latin binomial, including full Methanocaldococcus Whitman 2002 taxonomic authority: Methanococcus Kluyver and van Niel 1936 emend. Barker 1936 Methanotorris Whitman 2002

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanococci

Order: Methanococcales

Family: Methanocaldococcaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information, including presence of any inseparable or associated organisms and any related animals present in New Zealand:

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Latin binomial, including full Methanocella Sakai et al. 2008 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class:

Order: Methanocellales

Family: Methanocellaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Methanopyrales Huber and Stetter 2002 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanopyri

Order: Methanopyrales

Family Methanopyraceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

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Latin binomial, including full Methanospirillum Ferry et al. 1974 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanomicrobia

Order: Methanomicrobiales

Family Methanospirillaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Methanothermus Stetter et al. 1982 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Methanobacteria

Order: Methanobacteriales

Family Methanothermaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Eubacterium Prévot 1938 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Clostridia

Order: Clostridiales

Family Eubacteriaceae

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Strain(s) if relevant: Non pathogenic laboratory strains, – e.g. strain SA11

Other information

Latin binomial, including full Clostridium Prazmowski 1880 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Clostridia

Order: Clostridiales

Family Clostridium;

Strain(s) if relevant: Non pathogenic laboratory strains – e.g. strain CA6

Other information

Latin binomial, including full Blautia Liu et al. 2008 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Clostridia

Order: Clostridiales

unclassified Family Clostridiales;

Strain(s) if relevant:

Other information

Latin binomial, including full Oxobacter Collins et al. 1994 taxonomic authority:

Type of organism Microorganism

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Taxonomic class, order and family: Class: Clostridia

Order: Clostridiales

Family Clostridiaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Acetitomaculum Greening and Leedle 1995 taxonomic authority:

Type of organism Microorganism

Taxonomic class, order and family: Class: Clostridia

Order: Clostridiales

Family Lachnospiraceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information

Latin binomial, including full Brevibacillus choshinensis (Takagi et al. 1993), Shida taxonomic authority: et al. 1996

Type of organism Bacterium

Taxonomic class, order and family: Class: Bacilli

Order: Bacillales

Family Paenibacillaceae

Strain(s) if relevant: Non pathogenic laboratory strains

Other information This is a commercial strain available from Takara for protein expression. An essential gene for spore formation cascade is disrupted in B. choshinensis SP3.

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Latin binomial, including full Bacillus megaterium de Bary 1884 taxonomic authority:

Type of organism Bacterium

Taxonomic class, order and family: Class: Bacilli

Order: Bacillales

Family Paenibacillaceae

Strain(s) if relevant: Non pathogenic commercial strains for laboratory use, for example, strains available from MoBiTec for protein expression, such as: WH320 developed by Prof. Dr. W. Hillen at the Institute of Microbiology in Erlangen, Germany. MS941. Both strains are mutants of DSM319, where Other information WH320 is a chemically mutant, while MS941 has a defined deletion in the gene of the major extracellular protease NprM

Latin binomial, including full Bacillus subtilis (Ehrenberg 1835) Cohn 1872 taxonomic authority:

Type of organism Bacterium

Taxonomic class, order and family: Class: Bacilli

Order: Bacillales

Family Paenibacillaceae

Strain(s) if relevant: Non pathogenic commercial strains for laboratory use, for example, strains available from MoBiTec for protein expression, such as:

1012 wild type: leuA8 metB5 trpC2 hsdRM1 (commonly used) 168 Marburg: trpC2 (Trp - )

WB800N: nprE aprE epr bpr mpr :: ble nprB :: bsr .vpr wprA :: hyg cm :: neo; NeoR

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Other information WB800N carries resistance to neomycin

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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.

Escherichia coli Yes No

1 Is the organism normally capable of causing disease in humans, X animals, plants or fungi? If yes, provide details here 2 Is the organism a human cell line? X 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? X 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 X disease in humans, animals, plants or fungi? If yes, provide details here.

5 Does the organism produce desiccation resistant structures (such as X spores or cysts) that can normally be disseminated in the air? No. The essential gene for spore formation cascade is disrupted in B. choshinensis SP3 6 Is the organism characterised to the extent that its main biological X characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? X

If yes, provide details here.

8 If the organism is a whole plant or plant tissue, do you intend to: N/A N/A 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 X Genetic Modification) Regulations 2003)? 10 Is the host a Category 2 organism (as defined in the HSNO (Low-Risk X Genetic Modification) Regulations 2003)?

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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.

Brevibacillus choshinensis Yes No

5 Does the organism produce desiccation resistant structures (such as X 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 X characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? X

If yes, provide details here.

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Bacillus subtilis Yes No

5 Does the organism produce desiccation resistant structures (such as X spores or cysts) that can normally be disseminated in the air? Bacillus subtilis is a non-pathogenic spore-producing bacterium

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

If yes, provide details here.

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Bacillus megaterium Yes No

Is the organism normally capable of causing disease in humans, X animals, plants or fungi? If yes, provide details here Is the organism a human cell line? X 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) Is the organism native to New Zealand? X 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 Does the organism contain infectious agents normally able to cause X disease in humans, animals, plants or fungi? If yes, provide details here. Does the organism produce desiccation resistant structures (such as X spores or cysts) that can normally be disseminated in the air? Bacillus megaterium is a non-pathogenic spore-producing bacterium Is the organism characterised to the extent that its main biological X characteristics are known? Does the organism normally infect, colonise or establish in humans? X If yes, provide details here. If the organism is a whole plant or plant tissue, do you intend to: N/A N/A 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? Is the host a Category 1 organism (as defined in the HSNO (Low-Risk X Genetic Modification) Regulations 2003)? Is the host a Category 2 organism (as defined in the HSNO (Low-Risk X Genetic Modification) Regulations 2003)?

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Risk group 1 methanogen species from New Zealand ruminants belonging to the genus of:

Methanobrevibacter (Balch and Wolfe 1981) Methanobacterium(Kluyver and van Niel 1936) Methanobacter Wasserfallen et al. 2000 Methanohalobium Zhilina and Zavarzin 1988 Methanocalculus Ollivier et al. 1998 Methanocaldococcus Whitman 2002 Methanocellaceae Sakai et al. 2008 Methanococcus Kluyver and van Niel 1936 emend. Barker 1936 Methanococcoides Sowers and Ferry 1985 Methanocorpusculum Zellner et al. 1988 Methanoculleus Maestrojun et al. 1990 Methanofollis Zellner et al. 1999 Methanogenium Romesser et al. 1981 Methanohalophilus Paterek and Smith 1988 Methanolacinia Zellner et al. 1990 Methanolobus Konig and Stetter 1983 Methanomethylovorans Lomans et al. 2004 Methanomicrobium (Balch and Wolfe 1981) Methanimicrococcus corrig. Sprenger et al. 2000 Methanoplanus Wildgruber et al. 1984 Methanopyrales Huber and Stetter 2002 Methanosaeta Patel and Sprott 1990 Methanosalsum Boone and Baker 2002 Methanosarcina (Kluyver and van Niel 1936) Methanosphaera (Miller and Wolin 1985) Methanospirillum Ferry et al. 1974 Methanothermus Stetter et al. 1982 Methanotorris Whitman 2002

Risk group 1 acetogen species from New Zealand ruminants belonging to the genus of:

Eubacterium Prévot 1938 Blautia Liu et al. 2008 Oxobacter Collins et al. 1994 Acetitomaculum Greening and Leedle 1995

Risk group 1 methanogen and acetogen species isolated from Yes No New Zealand ruminants (as listed above)

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Committee (if required) 3 Is the organism native to New Zealand? X 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 X disease in humans, animals, plants or fungi? If yes, provide details here.

5 Does the organism produce desiccation resistant structures (such as X 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 X characteristics are known? 7 Does the organism normally infect, colonise or establish in humans? X

If yes, provide details here.

Risk group 1 Clostridium species isolated from New Zealand Yes No ruminants (as listed above)

Page 21 of 30 Develop in containment a project of low risk genetically modified organisms by rapid assessment needed to determine whether Māori have been consulted appropriately Does the organism contain infectious agents normally able to cause X disease in humans, animals, plants or fungi? If yes, provide details here. Does the organism produce desiccation resistant structures (such as X spores or cysts) that can normally be disseminated in the air? Some species of Clostridium are known to produce spores Is the organism characterised to the extent that its main biological X characteristics are known? Does the organism normally infect, colonise or establish in humans? X If yes, provide details here. If the organism is a whole plant or plant tissue, do you intend to: N/A N/A 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? Is the host a Category 1 organism (as defined in the HSNO (Low-Risk X Genetic Modification) Regulations 2003)? Is the host a Category 2 organism (as defined in the HSNO (Low-Risk X Genetic Modification) Regulations 2003)?

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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 commercially available bacterial cell expression, plasmid, or viral cloning vectors such as pGTC, pTrueBlue-rop, fosmids such as pCC1Fos, bacterial artificial chromosomes (BACs) such as pCC1BAC and expression vectors such as Gateway cloning system vectors. The methanogen gene manipulation vectors will be plasmids such as pKH, pPAC and pNPAC. These are Methanococcus voltae plasmid vectors that are based on the E. coli plasmid pUC and carry the bla gene encoding ampicillin resistance. They also carry the pac gene which encodes a puromycin transacetylase which confers resistance to puromycin. The pac gene is flanked by the promoter and terminator of the methyl CoM reductase (mcr) transcription unit from Methanococcus voltae which allows expression of puromycin resistance in methanogens. Naturally occurring plasmids such as pC2A from Methanosarcina acetovorans will also be used. Range of elements that the vectors Vectors will include standard and commercially may contain available promoters and other gene regulatory elements, reporter and selectable marker genes, protein purification tags and origins of replication.

Type, source and function of any Ruminant donor genetic material will include donor genetic material coding, non-coding or regulatory regions of genes involved in the synthesis and/or regulation of methane, acetate or other relevant biochemical pathways mediating methane mitigation strategies. Donor genetic material from other kingdoms that code for gene regulatory elements, reporters or selectable markers may be used.

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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.

If yes, provide details here including where the material is obtained from, and whether approval has been obtained from an Ethics Committee (if required). Also complete section 5 of this form.

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.

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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.

E. coli, B. choshinensis, and Risk Group 1 Methanogen and Yes No Acetogen Species

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

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

Will the proposed modification increase the pathogenicity, virulence, X 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 X 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 X 5 PC1 containment?

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

Does the proposed modification conform to the requirements of a X 7 Category A genetic modification?

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

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B. subtilis, B. megaterium and Risk group 1 Clostridium species Yes No

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

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

Is the proposed modification to be carried out under a minimum of X 5 PC1 containment?

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

Does the proposed modification conform to the requirements of a X 7 Category A genetic modification?

Does the proposed modification conform to the requirements of a X 8 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 MAF Biosecurity Authority/ERMA New containment facility approved under? Zealand Standard: Facilities for Microorganisms and Cell Cultures: 2007a

The Australian/New Zealand Standard 2243.3:2002 Safety in laboratories: Microbiological aspects and containment facilities, What physical containment level The experiments will be conducted under a (AS/NZS 2243.3:2002) is this minimum of PC1 level containment. All containment facility approved to experiments involving B. subtilis, B. megaterium operate at (where relevant)? and risk group 1 Clostridium species will be conducted under a minimum of PC2 level containment. What other physical measures do you Experiments with methanogens and acetogens propose to use to contain this will be carried out in sealed containers or within organism? an anaerobic chamber with a minimum containment level of PC1 for the non-spore- forming species, and PC2 for risk group 1 Clostridium species. What procedural or operational The experiments will be conducted by trained measures do you propose to use to staff working under a minimum of PC1 contain this organism? containment. All experiments involving B. subtilis, B. megaterium and risk group 2 Clostridium species will be conducted under a minimum of PC2 level containment. To minimise the chances of escape of organisms from the laboratory, all cultures and consumables used in experiments will be autoclaved on site prior to disposal. Any other information relevant to the Rumen methanogens and acetogens (including containment of the organism. rumen Clostridium species) are extremely sensitive to oxygen and require stringent conditions for growth, which represent additional barriers to escape from the laboratory

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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 AgResearch are secure and precautions (as described in section Four) are in place to prevent possible escape. Therefore no significant adverse effects have been identified. All microbial sources and hence genetic material is isolated from NZ ruminants and thus are already present in the NZ environment. What adverse effects could this organism have on human health and safety? No significant adverse effects have been identified.

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)? The development of the GMOs does not involve any native or human genetic material. Our local iwi representative, Mr Jonathan Proctor, of Tanenuiarangi Manawatu Inc. has reviewed this application and has not identified any significant adverse effects. Tanenuiarangi Manawatu Inc have no concerns with the application and work progressing. 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 GMD08020 this application previously been considered in New Zealand or 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 Y Depending on the success of our research organism what will the genetically programme, in the future we might conduct modified organism be used for? eg animal exposure studies using the will experimental animals or plants developed GMOs. We will obtain approval be exposed to this organism? from a relevant animal welfare ethics committee such as the Ruakura Animal Ethics Committee before these experiments are commenced. These experiments will be performed within a minimum indoor PC1 containment facility registered to the appropriate MAF Biosecurity Authority/ERMA New Zealand Standard to ensure that no GMOs escape and form a self-sustaining population

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

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.

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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.

List of appendices attached:

List of references attached:

Leahy SC, Kelly WJ, Altermann E, Ronimus RS, Yeoman CJ, et al. (2010) The Genome Sequence of the Rumen Methanogen Methanobrevibacter ruminantium Reveals New Possibilities for Controlling Ruminant Methane Emissions. PLoS ONE 5(1): e8926. doi:10.1371/journal.pone.0008926 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0008926

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