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. Page 2 of 28 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: 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. Page 3 of 28 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. 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. Page 4 of 28 Develop in containment a project of low risk genetically modified organisms by rapid assessment 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 micro- organisms, 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). Page 5 of 28 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 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.