application title: applicant organisation · application. you need to justify why you consider the...

NO3P

ER-AF-NO3P-3

12/07

Develop in containment a project of low risk genetically

modified organisms by rapid assessment

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

Application title: Cloning and Expression of Proteins from Extremophiles for

Biophysical and Biochemical Characterization.

Applicant organisation: Institute of Geological & Nuclear Sciences (GNS Science)

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

Confidential information identified and enclosed separately NA

Copies of references attached NA

Application signed and dated Yes

Electronic copy of application e-mailed to ERMA New

Zealand

Yes

Signed: Date: 26/11/2010

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

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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: Institute of Geological & Nuclear Sciences

Postal Address: PO Box 2000, Taupo, New Zealand. 3352

Physical Address: 114 Karetoto Rd., SH1, Wairakei, Taupo. 3377

Phone: (07) 374 8211

Fax: (07) 374 8199

Email:

Name and details of the key contact person (if different from above):

Name: Matthew Stott

Postal Address: As above

Physical Address: As above

Phone: As above

Fax: As above

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.

The Extremophile Research Group at the Institute of Geological & Nuclear Sciences (GNS Science) is undertaking research chiefly based around the discovery and investigation into novel New Zealand indigenous extremophilic microorganisms from their natural environments. Extremophilic organisms live in environments not normally inhabitable by the majority of life, such as bubbling hot springs, highly salty brine water, deep underground oil reserves, acidic or alkaline waters or near hydrothermal vents at the bottom of the ocean. New Zealand contains a diverse number of terrestrial, marine and deep biosphere “extreme” environments within the exclusive economic zone (EEZ) and it is anticipated that these sites may contain undiscovered microbial populations. We hope that by studying microorganisms that inhabit these environments, we will learn more about how these organisms are able to survive in these harsh conditions and how they interact with the food chain and surrounding geochemical/mineral environments.

Discovering new microbes also opens up possibilities of developing potentially new beneficial technologies such as bioremediation of pollutants, generation of environmentally sustainable energies, medicine and drug discovery, and efficient industrial processes. In order to develop these technologies, putative enzymes need to be expressed independently to determine their activity and/or their identity. This application proposes to investigate non-pathogenic, non-infectious and non-toxic genes of the putative enzymes from extremophilic microorganisms by genetically modifying non-pathogenic laboratory strains of Escherichia coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae.

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.

Purpose of the project: To genetically modify non-pathogenic strains of Escherichia coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae with genes from extremophilic microorganisms (see list of abbreviations for Extremophiles definition) in order to investigate the structure and function of proteins for the development of biofuels, new antibiotics, and/or molecular biology tools. In addition, putative genes encoding for proteins involved in general catabolic and anabolic metabolism of extremophiles will also be investigated to decipher function. All modifications will use routine biophysical and biochemical


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methodologies.

Aims: The aim of this project is to characterize proteins from a variety of extremophilic microorganisms using biophysical techniques and biochemical assays.

Description of GMOs to be developed: Escherichia coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae (non-pathogenic laboratory strains) will be modified to express genes from extremophilic organisms listed encoding proteins from the following classes: cell matrix proteins, cellulases, lignin-active enzymes, glycosyl transferases, antimicrobials, transcription factors, proteins involved in ATP synthesis and respiration, oxidoreductases, lipid biosynthesis proteins and cell-to-cell signalling proteins. Genes encoding these proteins will be sourced from the extremophilic bacterial species. Each of these protein classes are entirely nontoxic, nonpathogenic and incapable of causing disease. The function of these proteins is contained in the above listing. In the future we intend to amend this application (if it is approved) to include the development of a third, as-of-yet, undefined extremophilic GMO bacterial host. The rational behind this amendment will be that an extremophilic host will be more likely to be able to produce a functional “extremophilic” protein.

The genes will be cloned in non-pathogenic laboratory strains of E. coli, B.choshinensis and/or S. cerevisiae using standard expression vectors.

All genes will be either amplified from purified nucleic acid or obtained from GMOs containing plasmids which express the gene of interest imported under appropriate ERMA approvals.

The vector and donor nucleic acids to be introduced will include promoters and other gene regulatory elements, reporter and selectable marker genes, tagging sequences and origins of replication, but will not include any of the following:

genes encoding vertebrate toxins

sequences that will produce particles able to infect humans, animals or plants

modifications that will result in the GMO having a greater ability to escape from containment that the unmodified host organism

material from Māori

material from species listed by the Convention on International Trade in Endangered Species (CITES), except where accompanied by appropriate letters of approval from the relevant agencies in the exporting and importing countries.

uncharacterised nucleic acid sequences from pathogenic microorganisms material from native plant or animal species


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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 Escherichia coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae to express genes from extremophilic bacteria in order to produce proteins for biophysical and biochemical analysis.

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

taxonomic authority:

Escherichia coli (Migula 1895) Castellani and Chalmers 1919.

Common name(s), if any: Bacteria:

N/A

Type of organism (eg bacterium,

virus, fungus, plant, animal, animal

cell):

Bacterium;

Taxonomic class, order and family: Bacteria (domain); Proteobacteria (phylum); Gammaproteobacteria (class); Enterobacteriales (order); Enterobacteriaceae (family); Escherichia (genus); coli (species).

Strain(s) if relevant: Non-pathogenic Escherichia coli K-12 and B derivatives.

Other information, including

presence of any inseparable or

associated organisms and any related

animals present in New Zealand:

There are no known inseparable or associated organisms.


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Brevibacillus choshinensis

(Takagi et al. 1993) Shida et al. 1996

Common name(s), if any: Bacteria:

N/A



cell):

Bacterium;

Taxonomic class, order and family: Bacteria (domain); Firmicutes (phylum); Bacilli (class); Bacillales (order); Paenibacillaceae (family); Brevibacillus (genus); choshinensis (species).

Strain(s) if relevant: Non-pathogenic Brevibacillus choshinensis

SP3 strain, does not form spores








Yeast Saccharomyces cerevisiae (Meyen ex EC Hansen (1883)) (non pathogenic laboratory adapted strains)

Common name(s), if any: Yeast:

Baker‟s yeast



cell):

Bacterium; and

Yeast respectively

Taxonomic class, order and family: Fungi (domain); Ascomycota (phylum); Saccharomycetes (class); Saccharomycetales (order); Saccharomycetaceae (family); Saccharomyces (genus); cerevisiae


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(species)

Strain(s) if relevant: .






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,

animals, plants or fungi? X

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

disease in humans, animals, plants or fungi? X

If yes, provide details here.

5 Does the organism produce desiccation resistant structures (such as

spores or cysts) that can normally be disseminated in the air? X


6 Is the organism characterised to the extent that its main biological

characteristics are known? X

7 Does the organism normally infect, colonise or establish in humans? X



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8 If the organism is a whole plant or plant tissue, do you intend to:

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?

N/A

9 Is the host a Category 1 organism (as defined in the HSNO (Low-Risk

Genetic Modification) Regulations 2003)? X



Brevibacillus choshinensis SP3

Yes No

















This strain of Brevibacillus choshinensis has been modified not to

produce spores






a) Allow it to develop reproductive structures If yes, please


b) Keep it in a closed container?

N/A


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Saccharomyces cerevisiae

Yes No























c) Allow it to develop reproductive structures If yes, please


d) Keep it in a closed container?

N/A





The strains of Escherichia coli, Saccharomyces cerevisiae and Brevibacillus choshinensis are

a non pathogenic organisms and are classed as category 1 host organisms since it is—


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- is clearly identifiable and classifiable according to genus, species, and strain or other

sub-specific category as appropriate; and

- is not normally able to cause disease in humans, animals, plants, or fungi; and

- does not contain infectious agents normally able to cause disease in humans,

animals, plants, or fungi; and

- does not produce desiccation-resistant structures, such as spores or cysts, that can

normally be disseminated in the air; and

- is characterised to the extent that its main biological characteristics are known; and

- does not normally infect, colonise, or establish in humans

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


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

expression, plasmid, or viral

Standard, non-conjugative E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae cloning and expression plasmid vectors.

Range of elements that the vectors

may contain

Origin for replication in E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae, a multiple cloning site, selectable markers and regulatory elements for expression of target genes, tagging elements or reporter genes.

Type, source and function of any

donor genetic material

The donor genetic material will be sourced from extremophilic bacterial species. The donor genetic material will encode proteins from the following classes: cell matrix proteins, cellulases, lignin-active enzymes, glycosyl transferases, antimicrobials, transcription factors, proteins involved in ATP synthesis and respiration, oxidoreductases, lipid biosynthesis proteins and cell-to-cell signalling proteins.

Use of special genetic material

Yes No


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Does the proposed modification use genetic material derived from

organisms capable of causing disease in humans, animals, plants or fungi? X

Does the proposed modification use genetic material from native biota? X

Does the proposed modification involve human genetic material? Answer

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.

X

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.

Target genes encoding cellulases, ligninases, antimicrobial agents, general respiratory enzymes (including enzymes involved in anabolic and catabolic reactions), and/or associated proteins from biosynthesis operons will be amplified from plasmids using polymerase chain reaction (PCR) with specific primer oligonucleotides. PCR fragments will be inserted into the multiple cloning sites of standard E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae expression vectors. If subcloning of PCR fragments is necessary, standard cloning plasmids will be used. Non-pathogenic strains of E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae (all K12 or B derivatives) will be transformed with plasmid DNA using established heat shock or electroporation protocols. Expression of target proteins in non-pathogenic E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae cells will be accomplished by the use of IPTG or other inducible systems.


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3.4 Identify the category of experiments as described in the HSNO (Low-Risk

Genetic Modification) Regulations, 2003.


section.

Yes No

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

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

3 Will the proposed modification increase the pathogenicity, virulence,

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.

X

4 Will the proposed modification result in a genetically modified

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.

X

5 Is the proposed modification to be carried out under a minimum of

PC1 containment? X

6 Is the proposed modification to be carried out under a minimum of

PC2 containment? X

7 Does the proposed modification conform to the requirements of a

Category A genetic modification? X

8 Does the proposed modification conform to the requirements of a

Category B genetic modification? X

Explanation of categorisation, if necessary. This is particularly important for work

involving pathogenic microorganisms and viral vectors

E. coli strain K12, Brevibacillus choshinensis SP3 and/or Saccharomyces cerevisiae derivatives are category 1 hosts. They will be modified with characterised genes from bacteria that will not result in a host organism that is more pathogenic than a category 1 host organism. The modified organisms will not have a greater ability to escape from containment than the unmodified host organism. The modifications therefore meet the criteria for category A genetic modifications.


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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

containment facility approved under?

MAF /ERMA Regulatory Standard Facilities for Microorganisms and Cell Cultures: 2007a

What physical containment level

(AS/NZS 2243.3:2002) is this

containment facility approved to

operate at (where relevant)?

PC1.

What other physical measures do you

propose to use to contain this

organism?

N/A

What procedural or operational

measures do you propose to use to

contain this organism?

The work will be carried out under a minimum of PC1 conditions as described in AS/NZS 2243.3. Procedures prescribed in the Facility Containment Manual will be used to ensure effective containment (attached). While the facility is officially classed as a PC1 facility, the laboratory operate at levels equivalent to PC2 including sterilizing all materials in contact with biological material, locked laboratories and appropriate culturing conducted in a Biohazard Containment Hood

Any other information relevant to the

containment of the organism. The Wairakei facility has a MAF Containment facility reference number of 846.


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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

This project employs debilitated E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae strains that have strict nutritional requirements and would be unable to establish a self-sustaining population if they escape from containment and therefore there is a minimal risk of adverse effects on the environment.

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

This project employs debilitated E. coli, Brevibacillus choshinensis and/or Saccharomyces cerevisiae strains that have strict nutritional requirements that are unable to colonise humans and therefore there is a minimal risk of adverse effects on human health and safety.

What adverse economic effects could this organism have?

No adverse economic effects from this contained non-pathogenic GMO have been identified.


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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)?

All work using microorganisms enriched from Māori landholdings and ancestral lands are conducted in full knowledge, guidance and permission of the landowners. Specifically, microorganisms, and sediments that may contain microorganisms, have be collected from geothermal areas (hotsprings, mudpools, fumaroles, etc) in the Taupo Volcanic Zone (TVZ) and from hydrothermal vents from the Kermadec Arc. Scientific work conducted by researchers at the Institute of Geological & Nuclear Sciences (GNS Science) in the Taupo Volcanic Zone has been conducted since 2001 and has been covered by Department of Conservation (DOC) Collecting Permits and via permission for local Māori guardians. During this time a good working relationship has been built between local Iwi and GNS Science. GNS has a legal and sampling agreement with Tikitere Trust that covers research and benefit sharing (see appendices 1 for scan of the title page of the Agreement), and is currently in negotiation with Ngati Tahu Ngati Whaoa for a similar agreement.

The DOC permitting process requires that applications to be reviewed by the local Māori guardians. During this process we have had enquiries on the objectives of our scientific research and have explained the type of research, the type of samples to be collected, and the long term objectives of the research.

All ERMA, MAF, MFISH and DOC risk assessments and permits for samples collected from the Kermadec Arc and terrestrial TVZ have been fulfilled.

Are there any other potential adverse effects?

There are no other anticipated adverse effects.


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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

approvals under any other New

Zealand legislation?

N

Does New Zealand have any

international obligations relating to

(any of) the organism(s)?

N

Have any of the new organism(s) in

this application previously been

considered in New Zealand or

elsewhere?

Y GMD05027

NOC06002 would be used by the commercial commonly supplying the host organism Brevibacillus choshinensis SP3

Is there any additional information

that you consider relevant to this

application that has not already been

included?

N

Following the development of this

organism what will the genetically

modified organism be used for? eg

will experimental animals or plants

be exposed to this organism?

Organisms will be kept in laboratory culture collections and used solely for the purpose of maintaining plasmid DNA and/or producing proteins of interest.

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

Antimicrobial agents: proteins or chemicals generated by microorganisms that kill or inhibit other microorganisms. These may include restriction enzymes (enzymes that cleave DNA at defined sequences), antibiotics, and/or lantibiotics (a protein version of an antibiotic).

Cellulases : enzymes involved in the hydrolysis of cellulose or hemicellulose polymers

Extremophilic microorganisms require „extreme‟ physio-chemical conditions for optimal growth. These conditions are usually outside those that permit the majority of life to exist;

o acidophiles (acidity pH <3.0) o thermophiles (Temperatures >45˚C) o hyperthermophiles (Temperatures >80˚C) o methanogens (microorganisms that generate methane and are strict

anaerobes) o barophiles (requirement of a atmospheric pressure greater than 1atm for

growth) o halophiles (requirement of a minimum NaCl requirement, usually

considered greater than that of seawater)


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Hydrothermal vent/feature; Hydrothermal vents arise from seawater being heated by magmatic features near to the earths surface. Hydrothermal vents are usually associated with marine systems and are most readily located is the vicinity of tectonic plate edges.

Lignin-active enzymes: enzymes involved in the hydrolysis of lignin and its components including peroxidises, laccases and dioxygenases.

NZ EEZ; New Zealand Exclusive Economic Zone

Operon: a cluster of genes under the control of a single promoter. Generally, these genes encode for similar or related functions.

Oxidoreductases: an enzyme that catalyses the transfer of electrons between compounds.

Respiration: the process of converting biochemical energy from nutrients to usable cellular (usually in the form of ATP – adenosine triphosphate), and then releasing waste products.

Transcription factor: a protein that binds to DNA and controls transcription.

TVZ: Taupo Volcanic Zone.


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List of appendices attached:


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List of references attached:

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