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. Page 2 of 30 Develop in containment a project of low risk genetically modified organisms by rapid assessment 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. Page 3 of 30 Develop in containment a project of low risk genetically modified organisms by rapid assessment 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. Page 4 of 30 Develop in containment a project of low risk genetically modified organisms by rapid assessment 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. Page 5 of 30 Develop in containment a project of low risk genetically modified organisms by rapid assessment 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