APPLICATION FORM Containment – GMO Project
To obtain approval for projects to develop genetically modified organisms in containment
Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.
Application Number
GMO17LU001
Date
26 June 2017
www.epa.govt.nz 2
Application Form Approval for projects to develop genetically modified organisms in containment
Completing this application form
1. This form has been approved under section 42A of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers projects for development (production, fermentation or regeneration) of genetically modified organisms in containment. This application form may be used to seek approvals for a range of new organisms, if the organisms are part of a defined project and meet the criteria for low risk modifications. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 2. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise. Commercially sensitive information
10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available.
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Application Form Approval for projects to develop genetically modified organisms in containment
12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions
Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation
Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment
Any organism in which any of the genes or other genetic material: Have been modified by in vitro techniques, or Genetically Modified Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques
A new organism is an organism that is any of the following: An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation; An organism for which a containment approval has been given under the HSNO Act; An organism for which a conditional release approval has been given under the HSNO Act; New Organism A qualifying organism approved for release with controls under the HSNO Act; A genetically modified organism; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand; An organism present in New Zealand before 29 July 1998 in contravention of the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because: It is subject to a conditional release approval; or It is a qualifying organism approved for release with controls; or It is an incidentally imported new organism
An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal
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Application Form Approval for projects to develop genetically modified organisms in containment
1. Applicant details
1.1. Applicant
Company Name: (if applicable) Lincoln University
Contact Name: Jon Hickford
Job Title: Professor
Physical Address: Ellesmere Junction Road, Lincoln, Canterbury
Postal Address (provide only if not the same as the physical):
Phone (office and/or mobile): 03 423 0665
Fax:
Email: [email protected]
1.2. New Zealand agent or consultant (if applicable)
Company Name:
Contact Name:
Job Title:
Physical Address:
Postal Address (provide only if not the same as the physical):
Phone (office and/or mobile):
Fax:
Email:
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Application Form Approval for projects to develop genetically modified organisms in containment
2. Information about the application
2.1. Brief application description Approximately 30 words about what you are applying to do
Application to genetically modify local isolates of bacteria Brevibacillus laterosporus, by disrupting genes thought to be associated with sporulation and toxin production to establish if these genes influence the known insecticidal activity of this biocontrol agent.
2.2. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it
As part of a larger research programme on potential bio-control agents to control insect pests in cropping systems, investigations into a potential bio-control agent against the diamondback moth (DBM) are being undertaken.The potential agents are New Zealand isolates of the bacterium Brevibacillus laterosporus. The DBM is an established insect pest in New Zealand and is also a major pest in various parts of the world (Sarfraz et al., 2005). It feeds mainly on brassica species such as cabbage, cauliflower and broccoli, causing substantial damage to crops and affecting brassica growers throughout the country. Several B. laterosporus bacterium isolated in New Zealand show high insecticidal activity against the DBM caterpillars. The bacterium is similar in various ways to the related Bacillus thuringiensis (Bt) which is used as a bio-control agent. However, some DBM populations are starting to show significant resistance towards Bt toxins (Bravo & Soberón, 2008), which prompted the need to find for alternatives.
Our research has demonstrated that B. laterosporus produces toxins capable of killing DBM caterpillars. In order to maximize the potential of B. laterosporus as a biocontrol agent, our aim is to understand how the insecticidal activity of B. laterosporus is controlled. We aim to inactivate genes thought to be involved in the insecticidal acitivty and then monitor resultant insecticidal ability of the bacteria. Insecticidal activity of these genetically modified B. laterosporus will be determined by exposing insects (mainly DBM caterpillars) to preparations of cell-free culture medium, pelleted bacterial spores and any associated spore components, derived from the growth of these altered microbes under strictly controlled laboratory conditions. This study is expected to result in a reduction of insecticidal activity.
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Application Form Approval for projects to develop genetically modified organisms in containment
2.3. Technical description
Briefly describe the host organism(s) and the proposed genetic modifications. Please make sure that any technical words used are included in a glossary. Note if any part of this research project is already covered by an existing HSNO Act approval that your organisation holds or uses.
Brevibacillus laterosporus is a spore-forming bacillus which is found in water and soil world-wide, including in New Zealand. They are of particular interest as some strains are known to kill insects, nematodes and molluscs and therefore have potential as bio-control agents. Pathogenic effects on other plants or animals, including humans, have not been reported so far. Our own strains have been tested against small mammalians with no effect. Phylogenetic analysis of 16s rRNA sequences of the Bacillus genus showed that Brevibacillus laterosporus is far removed from human pathogens including Bacillus anthracis and Bacillus cereus. One strain of B. laterosporus has even been commercialised in the United States to improve human health as a probiotic.
In a Ministry of Business Innovation and Employment (MBIE) project to search for potential new microbes to control pests of brassica crops, isolates of B. laterosporus were recovered from the seed surface of brassicas. In simple bioassays these isolates were shown to kill DBM larvae. Furthermore, the insecticidal activity appeared to be associated with sporulation, suggesting that if the sporulation process can be influenced, so can insecticidal activity.
In the genomes of the NZ B. laterosporus are numerous homologs of previously characterised insect toxin genes. Under a previous approval (GMD100643), we cloned and expressed the cry toxins from these strains and found the proteins are not responsible for the DM toxicity. Therefore, a different approach is required to identify the processes involved in insecticidal activity. Genes expected to be involved are an identified surface-layer (S-layer) type protein, the toxin and virulence gene homologues and sporulation genes. None of the target toxin producing genes identified have any similarity to human toxin genes such as those produced by B. anthracis. Therefore study of the toxin genes does not pose a risk to human health or the environment.
As the toxin genes are located within the bacterial chromosome, there is no risk of transfer of the antimicrobial resistant genes via lateral transfer. Sporulation genes are located on the plasmid of B. laterosporus, however the expression of antimicrobial resistance genes increases the metabolic load on the bacteria and therefore would be less fit within the environment in the unlikely event of an escape from containment.
Our project is to genetically identify and disrupt genes involved in B. laterosporus sporulation and insecticidal toxin production. Thus, two biological systems will be used:
- Chemically competent E. coli cells will be transformed using the standard transformation method by heat shock.
- The final host bacterium B. laterosporus will be transformed by both standard chemical transformation by Tris-PEG, and electroporation.
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B. laterosporus-modified isolates will be phenotypically characterised to identify the effect of gene disruption on sporulation. In order to determine whether these genes could affect the production of molecular active molecules, these isolates will be used in in vivo bioassay against DBM (Section 4.2).
3. Information about the new organism(s)
3.1. Identity of the host organism(s)
For each host organism: Provide its taxonomic name and describe what type of organism it is. Provide a description of the strain(s) being applied for (if relevant). If the host organism is derived from humans (eg, cell lines) or may have cultural significance (e.g. sourced from native biota), provide details of its source. State the category (Category 1 or Category 2) of the host organism (as per the HSNO (Low Risk Genetic Modification) Regulations).
Latin binomial, including full taxonomic authority: Escherichia coli (Miguela, 1895)
Type of organism: Bacterium
Taxonimic class, order and family: Bacteria; Proteobacteria; Gammaproteobacteria;
Enterobacteriales; Enterobacteriaceae; Escherichia
Application of the strain(s) (if relevant): K12 derived strains
Is the organism derived from humans? Cultural significance No
(e.g. sourced from native biota):
Catergory of the host organism, as per HSNO (Low Risk Category 1
Genetic Modification Regulations):
Latin binomial, including full taxonomic authority: Brevibacillus laterosporus (Laubach, 1916; Shida, 1996)
Type of organism: Bacterium
Taxonimic class, order and family: Bacteria; Firmicutes; Bacilli; Bacillales; Paenibacillaceae;
Brevibacillus
Application of the strain(s) (if relevant):
Is the organism derived from humans? Cultural significance No
(e.g. sourced from native biota):
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Catergory of the host organism, as per HSNO (Low Risk Category 2
Genetic Modification Regulations):
3.2. Regulatory status of the organism
Is the organism that is the subject of this application also the subject of:
An innovative medicine application as defined in section 23A of the Medicines Act 1981?
☐ Yes ☒ No
An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?
☐ Yes ☒ No
4. Information about the project
4.1. Describe the nature and range of the proposed genetic modifications
Describe the nature and range of the proposed genetic modifications (e.g. the range of elements that the vectors or gene constructs may contain, and the type, source and function of the donor genetic material). State the category (Category A or Category B) of the genetic modifications (as per the HSNO (Low Risk Genetic Modification) Regulations).
The design of the gene-disruption plasmids will be based on integration vectors able to replicate in E. coli but not in gram-positive bacteria, such as B. laterosporus. pUC19 and pBR322 based plasmids are commonly used for this purpose and contains an origin of replication, a selectable marker and a multiple cloning site.
This non-conjugative vector will be coupled with counter-selectable and selectable markers:
- Under appropriate growth conditions a counter-selectable, such as lacZ, promotes the identification of plasmid-containing cells. In this case, selection is based on the production of colourless colonies due to insertional inactivation of the beta-galactosidase enzyme by the targeted gene.
- Antibiotics such as, ampicillin, tetracycline and chloramphenicol, are the most common selectable markers.
Homology-driven plasmid integration will be targeted to the specific locus on the chromosome or plasmid by homologous recombination and targeted gene will be disrupted by the cassette of the selectable
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The modifications to E. coli and B. laterosporus conform to Category A and B class regulations, respectively, as per the HSNO (Low Risk Genetic Modification) Regulations.
4.2. Proposed containment of the new organism(s) (physical and operational)
State which Containment Standard(s) your facility is approved to. State the minimum containment level (PC1 or PC2 as per AS/NZS2243.3:2002) required to contain the GMOs (as per the HSNO (Low Risk Genetic Modification) Regulations). Discuss whether controls in addition to the requirements listed in the Standard(s) are necessary to adequately contain the GMOs.
All work will be carried out in the Lincoln University Containment Facility (MPI 489) which is approved to standard 154.03.02 (Facilities for Microorganisms and Cell cultures:2007a), 154.02.17 (Transitional Facilities for Biological Products), 155.04.09 (Containment Facilties for Plants:2007) and 154.03.03 (Containment Facilties for Vertebrate laboratory animals) at a physical containment level of PC2 (AS/NZS2243.3:2002) and in accordance with the Lincoln University Quarantine and Containment Manual (V2). In order to assess the effect of deletion of targeted genes on sporulation and pathogenicity, insects such as Plutella xylostella larvae (Diamondback moth) will be tested for larval mortality. Following a modified protocol described by Shelton et al. (1993), cabbage leaf discs will be dipped in bacterial broth or supernatant obtained from modified B. laterosporus isolates. Early instar DBM larvae will be placed on each leaf disc inside screw-lid sealed vessels. Larval mortality will be assessed daily for at least 4 days after inoculation using a stereo microscope. All vessels will be clearly labelled and these insect assays will be carried out in closed incubators within the PC2 laboratory. Dead insect cadavers containing GM isolates will then be autoclaved within their vessels (a maximum of 7 days post-exposure, or prior to larvae maturation). All instruments used to handle insects and leaf discs will be disposed of by placing in paper envelopes and bagged into double lined autoclave waste bags in accordance with the Lincoln University Quarantine and Containment Manual (V6) prior to autoclaving. Instruments such as metal forceps will be re-used after sterilisation via autoclaving. Other instruments, such as plastic inoculation loops, will be disposed of after autoclaving.
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5. Risks, costs and benefits
Provide information of the risks, costs and benefits of the GMOs in the following areas of impact: The environment. Human health and safety. The economy (e.g. the ability of people and communities to provide for their economic wellbeing). The relationship of Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga, and the principles of the Treaty of Waitangi (The details of any engagement or consultations with Māori that you have undertaken in relation to this application should be discussed here). Society and community. New Zealand’s international obligations.
The B. laterosporus isolates are under development as active agent(s) in biopesticides, for use against brassica pests. Other B. laterosporus are also under development for use in biopesticides in Europe (Boets et al., 2011; Floris et al., 2010). Brevibacillus laterosporus has never been implicated in human disease or as a health risk.
The modifications proposed are unlikely to produce isolates with any increase in pathogenicity or ability to escape from containment. We are unaware of any Māori cultural significance attached to B. laterosporus and this work will not compromise any international obligations New Zealand may have.
If a biopesticide can be produced based on B. laterosporus it could be hugely benefical to New Zealand and the world, as a control product for pest insects. To accomplish this, data on the specific action of these genes is required, for regulators and patenting purposes, as well as for selection and production. If the S-layer protein is shown to be a toxin for insects, this would be novel and could help in development of novel biopesticides.
6. Other information
Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.
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7. Checklist This checklist is to be completed by the applicant
Application Comments/justifications All sections of the application form completed ☐ Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)
Confidential data as part of a separate, ☐ Yes ☐ No identified appendix
Supplementary optional information attached:
Copies of additional references ☐ Yes ☐ No
Relevant correspondence ☐ Yes ☐ No
Administration Are you an approved EPA customer? ☐ Yes ☐ No If Yes are you an: Applicant: ☐ Agent: ☐
If you are not an approved customer, payment of fee will be by: Direct credit made to the EPA bank ☐ Yes ☐ No account (preferred method of payment) ☐ Payment to follow Date of direct credit:
Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow
Electronic, signed copy of application e- ☐ Yes mailed to the EPA
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Signature of applicant or person authorised to sign on behalf of applicant
☒ I am making this application, or am authorised to sign on behalf of the applicant or applicant organisation.
☐ I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.
12 June 2016
Signature Date
Request for information waiver under section 59 of the HSNO Act
I request for the Authority to waive any legislative information requirements (i.e. concerning ☐ the information that has been supplied in my application) that my application does not meet (tick if applicable).
Please list below which section(s) of this form are relevant to the information waiver request:
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Appendices and referenced material (if any) and glossary (if required)
Glossary
Beta-galactosidase: Cleaves lactose in to glucose and galactose.
Bioassay- the use of a live test organism to determine the biological activity of a substance
Bio-control: Controlling pests, such as insects, weeds or plant disease using other organisms.
Chemically competent cells: Are bacterial cells treated with calcium chloride to facilitate the attachment of plasmid DNA to the bacterial cell membrane. Heating of the competent cells opens the pores of the cell membrane, allowing entry of the plasmid DNA.
Chromosome: A DNA molecule with part or all of the genetic material (genome) of an organism.
DNA: Deoxyribonucleic acid. The genetic material within almost all organisms.
Gene: A unit of heredity which is transferred from a parent to offspring, conferring a characteristic in the offspring.
Genome: The inherited genetic material of an organism.
Homology (sequence): The similarity in gene, DNA, or protein sequence.
Homologous recombination: Genetic recombination in which nucleotide sequences are exchange between two similar or identical DNA molecules.
Insecticidal: Destroying or controlling insects.
In vivo: Performed or taking place in a living organism.
Lac-Z: A gene within the lactose (lac) operon required for the metabolism of lactose in Escherichia coli. The Lac-Z protein is beta-galactosidase.
Multiple cloning site: A short segment of DNA which contains about 20 restriction sites. A standard feature of engineered plasmids.
Phenotype: The set of observable characteristics or traits, such as development, biochemical or physiological properties.
Phylogenetic: The study of the evolutionary history and relationships among individuals or groups of organisms. Methods analyse observed heritable traits, such as DNA sequences.
Plasmid: A circular DNA molecule that is separate and can replicate independently from the chromosomal DNA, and naturallay occurs in bacteria. Although it is sometimes found in eukaryotes as well.
Protein fractionation: A process or a series of processes intended to isolate a single or multiple type of protein from a complex mixture of proteins.
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Recombination: The exchange of genetic material either between different chromosomes or between different regions of the same chromosome.
Spore: A resistant form adopted by a bacterial cell in adverse conditions.
Sporulation: The formation of nearly dormant forms of the bacteria under unfavourable conditions.
Surface-layer protein: A protein attached to the bacterial cell wall. They have diverse functions including protection against environmental factors.
Transcription: The process wherein a DNA sequence is translated into a complementary sequence of messenger RNA (mRNA).
Tris-PEG: Tris (peg-2 phenylalanylcarboxamido) cyclohexane. An emulsion stabilising, viscosity controlling agent.
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References Boets, A., Arnaut, G., van Rie, J., & Damme, N. (2011). Belgium Patent No. US 7919609B2. U. S. P. a. T. Office. Bravo, A., & Soberón, M. (2008). How to cope with insect resistance to Bt toxins. Trends in Biotechnology, 26, 573-579. Floris, I., Ruiu, L., Satta, A., Delrio, G., Rubino, S., Paglietti, B., Pantaleoni, R. A. (2010). Italy Patent No. US 2010/0003227A1. U. S. P. a. T. Office. Sarfraz, M., Keddie, A. B., & Dosdall, L. M. (2005). Biological control of the diamondback moth, Plutella xylostella: A review. Biocontrol Science and Technology, 15, 763-789. Shelton, A. M., Robertson, J. L., Tang, J. D., Perez, C., Eigenbrode, S. D., Preisler, H. K., Wilsey, W. T., &Cooley, R. J. (1993). Resistance of diamondback moth (Ledipoptera:Plutellidae) to Bacillus thuringiensis subspecies in the field. Journal of Economic Entomology, 86,697-705.
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