NO3P
ER-AF-NO3P-3
12/07
Develop in containment a project of low risk genetically
modified organisms by rapid assessment
Application title: Expression of enzymes in E. coli and M. smegmatis
Applicant organisation: University of Canterbury
Considered by:
IBSC
ERMA
X
Please clearly identify any confidential information and attach as a separate appendix.
Please complete the following before submitting your application:
All sections completed
Appendices enclosed
Confidential information identified and enclosed separately
Copies of references attached
Application signed and dated
Electronic copy of application e-mailed to ERMA New
Zealand
Signed:
20 Customhouse Quay
Cnr Waring Taylor and Customhouse Quay
PO Box 131, Wellington
Phone: 04 916 2426 Fax: 04 914 0433
Email: info@ermanz.govt.nz
Website: www.ermanz.govt.nz
Date:
Yes
Yes/NA
Yes/NA
Yes/NA
Yes
Yes
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, crossreferenced, 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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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:
University of Canterbury
Postal Address:
School of Biological Sciences
University of Canterbury
Private Bag 4800
Christchurch
Physical Address:
School of Biological Sciences
University of Canterbury
Forestry Rd
Christchurch
Phone:
(03) 364 2987 x 7302
Fax:
(03) 364 2590
Email:
juliet.gerrard@canterbury.ac.nz
Name and details of the key contact person (if different from above):
Name:
Grant Pearce
Postal Address:
School of Biological Sciences
University of Canterbury
Private Bag 4800
Christchurch
Physical Address:
Phone:
(03) 364 2987 x 4848
Fax:
(03) 364 2590
Email:
grant.pearce@canterbury.ac.nz
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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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 nontechnical language.
Enzymes are proteins that catalyse (accelerate) chemical reactions. Almost all biological
processes rely on enzymes, as they allow reactions to occur at significant rates. An
understanding of how enzymes work is important in determining the metabolic pathways
that occur in different organisms. An example of this is the lysine biosynthesis pathway,
which occurs in plants and bacteria. Lysine is an essential amino acid, meaning that it is
only produced by plants and bacteria, and that animals obtain it through their diet.
Compounds that block the enzymes of the pathway may be able to be used as antibiotics
or herbicides.
Another example is the peroxiredoxin antioxidant defence pathway, which occurs in
plants, bacteria, fungi and mammals. Peroxiredoxins are an essential enzyme family that
break down peroxide, which is toxic to the cells at a high concentration. Study of these
enzymes may let us understand better the correlation between the activity and the
oligomeric state, and seek the potential for the oligomers to be applied in nanotechnology field.
In order to develop such inhibitors, we need to be able to first purify enzymes that are
potential drug or herbicide targets. If required, we may also express proteins (such as
chaperone proteins) to help the enzymes fold correctly. An example of a protein that is a
potential drug targets is Dihydrodipicolinate synthase from Mycobacterium tuberculosis.
Proteins can also be used for bioremediation, in which enzymes are used to break down
toxic substances, such as herbicides and pesticides that contaminate the environment. An
example of this is the use of phosphotriesterase from Pseudomonas diminuta to break
down organophosphates found in insecticides.
To do this, we use bacterial cells to produce sufficient amounts of protein, so that we can
look at the biochemical characteristics of the enzymes. The bacterial cells we use are
non-pathogenic laboratory strains of E. coli or M. smegmatis that have been modified so
that it is unlikely that these cells could survive outside of the lab environment.
The proteins that we express are not toxic to plants or animals, and are sourced from
mammals, bacteria, fungi, plant virus and plants that are not native to New Zealand.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
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 Project: To express enzymes in E. coli or M. smegmatis for subsequent
biochemical characterisation.
Description of GMOs to be developed: Genes encoding bacterial, fungal, plant viral and
plant enzymes will be expressed in non-pathogenic laboratory strains of E. coli or
M. smegmatis for the purposes of protein purification. Expressed protein will be
purified, in order to assay the biochemical function of the enzyme.
The genes will be derived from non-native bacteria, fungi, plant viruses and plants
and mammals (including humans cell lines), and do not encode any uncharacterised
nucleic acid sequences, vertebrate toxins, or factors that result in an increase in
pathogenicity, infectivity or virulence of the E. coli or M. smegmatis host (see
Appendix for examples of genes and donor sources).
The genes will be from enzymes or proteins (such as chaperone proteins) to help the
enzymes fold correctly.
No infectious particles will be produced. Proteins will only be expressed from
standard, non-conjugative expression bacterial plasmids, with no modification of the
host E. coli or M. smegmatis genome.
The standard cloning plasmid vectors containing the gene may contain promoter,
operator, regulatory element binding, expression tags, and selectable marker genes
that confer antibiotic resistance. Some modification of the expressed gene may be
carried out by site-directed mutagenesis, but there is negligible risk that these
modifications will result in an increase in toxicity, pathogenicity, infectivity or
virulence of the E. coli or M. smegmatis host.
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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 non-pathogenic laboratory strains of Escherichia coli or
Mycobacterium smegmatis to produce enzymes that are drug or herbicide targets, or for
bioremediation, to investigate their biochemical characteristics.
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 &
Chalmers, 1919) non-pathogenic laboratory
strains
Common name(s), if any:
E. coli
Type of organism (eg bacterium,
virus, fungus, plant, animal, animal
cell):
Bacterium
Taxonomic class, order and family:
Enterobacteriaceae
Strain(s) if relevant:
Commercially available non-pathogenic
laboratory strains
Other information, including
presence of any inseparable or
associated organisms and any related
animals present in New Zealand:
Strains do not contain conjugative plasmids or
generalised transducing phages
These strains have previously been approved
under our GMD08007 permit.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Latin binomial, including full
taxonomic authority:
Mycobacterium smegmatis (Trevisan 1889)
Lehmann and Neumann 1899 non-pathogenic
laboratory strains
Common name(s), if any:
M. smegmatis
Type of organism (eg bacterium,
virus, fungus, plant, animal, animal
cell):
Bacterium
Taxonomic class, order and family:
Actinomycetales, Mycobacteriaceae
Strain(s) if relevant:
Commercially available non-pathogenic
laboratory strains, such as 1-2c or mc2155
Other information, including
presence of any inseparable or
associated organisms and any related
animals present in New Zealand:
Strains do not contain conjugative plasmids or
generalised transducing phages.
Strains have previously been approved under
GMD07008
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
3.2
Information on the host organism
Complete this section separately for each host organism to be modified.
Escherichia coli
Yes
1
Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
No
No
If yes, provide details here
2
Is the organism a human cell line?
No
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?
No
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?
No
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?
No
If yes, provide details here.
6
Is the organism characterised to the extent that its main biological
characteristics are known?
7
Does the organism normally infect, colonise or establish in humans?
Yes
No
If yes, provide details here.
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?
NA
9
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Yes
10
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Page 8 of 16
NA
No
Develop in containment a project of low risk genetically modified organisms by rapid assessment
M. smegmatis
Yes
1
Is the organism normally capable of causing disease in humans,
animals, plants or fungi?
No
No
If yes, provide details here
2
Is the organism a human cell line?
No
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?
No
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?
No
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?
No
If yes, provide details here.
6
Is the organism characterised to the extent that its main biological
characteristics are known?
7
Does the organism normally infect, colonise or establish in humans?
Yes
No
If yes, provide details here.
8
If the organism is a whole plant or plant tissue, do you intend to:
c) Allow it to develop reproductive structures If yes, please
provide further information on containment in section 4
d) Keep it in a closed container?
NA
9
Is the host a Category 1 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
Yes
10
Is the host a Category 2 organism (as defined in the HSNO (Low-Risk
Genetic Modification) Regulations 2003)?
NA
No
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.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
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
E. coli and M. smegmatis
Vector system used, eg cloning or
expression, plasmid, or viral
Standard non-conjugative cloning and expression
plasmids (eg pET (Novagen), pBluescript
(Stratagene))
Range of elements that the vectors
may contain
The plasmid vectors containing the gene may
contain the following elements: promoter,
operator, regulatory element, expression tags, and
selectable marker genes that confer antibiotic
resistance.
Type, source and function of any
donor genetic material
Genetic material will be derived from non-native
bacteria, fungi, plant viruses and plants and
mammals, including human cell lines.
The genetic material will be from enzymes that
are potential drug or herbicide targets, have
potential in bioremediation, and proteins (such as
chaperone proteins) to help the enzymes fold
correctly
The genetic material will code for enzymes only,
and will not encode any vertebrate toxins, or any
factors that result in an increase in pathogenicity,
infectivity or virulence of the E. coli or M.
smegmatis host (see Appendix for examples of
genes and donor sources).
Only genes of known or putative enzymes will be
used. No uncharacterised sequences will be
expressed.
No infectious particles will be produced.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Use of special genetic material
Yes
Does the proposed modification use genetic material derived from
organisms capable of causing disease in humans, animals, plants or fungi?
No
Yes
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
See Appendix for examples of genes and donor sources.
Only genes of known or putative enzymes will be used. No uncharacterised
sequences will be expressed.
No infectious particles will be produced. No vertebrate toxins will be
expressed.
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
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.
Yes
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.
Human genetic material will not be derived directly from humans for this
study, hence ethical approval is not required. The material will be sourced
from the University of Otago cell line stocks, which are covered by
GMD002066 approval. Human genetic material will not be derived from
persons of Maori descent.
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.
Only standard molecular biology techniques will be used eg, PCR, DNA ligation, cloning,
transformation.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
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.
Yes
1
2
3
4
Is the proposed modification to a Category 1 host organism?
No
Yes
Is the proposed modification to a Category 2 host organism?
No
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.
No
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.
No
5
Is the proposed modification to be carried out under a minimum of
PC1 containment?
6
Is the proposed modification to be carried out under a minimum of
PC2 containment?
7
Does the proposed modification conform to the requirements of a
Category A genetic modification?
8
Does the proposed modification conform to the requirements of a
Category B genetic modification?
Yes
No
Yes
Explanation of categorisation, if necessary. This is particularly important for work
involving pathogenic microorganisms and viral vectors
Not applicable
Page 12 of 16
No
Develop in containment a project of low risk genetically modified organisms by rapid assessment
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-registered facilities that are approved under
the MAF/ERMA New Zealand Standard:
Facilities for Microorganisms and cell cultures:
2007.
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?
All users have been trained under AS/NZS
Standard 2243.3 (2002) Safety in Laboratories.
Part 3: Microbiological aspects and containment
facilities and the MAF/ERMA New Zealand
Standard: Facilities for Microorganisms and cell
cultures: 2007a.
What procedural or operational
measures do you propose to use to
contain this organism?
All users have been trained under AS/NZS
Standard 2243.3 (2002) Safety in Laboratories.
Part 3: Microbiological aspects and containment
facilities and the MAF/ERMA New Zealand
Standard: Facilities for Microorganisms and cell
cultures: 2007.
Any other information relevant to the
containment of the organism.
No
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
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
Should the non pathogenic laboratory stains of E. coli or M. smegmatis breach
containment, they are unlikely to establish a self-sustaining population, as the requirements
for essential amino acids and vitamins mean that they are unlikely to survive out of culture.
What adverse effects could this organism have on human health and safety?
Non pathogenic laboratory strains of E. coli or M. smegmatis are classified as Risk 1
organisms, and are unlikely to cause human, plant or animal disease, particularly as the
proposed modifications will not introduce traits to increase environmental fitness.
What adverse economic effects could this organism have?
We cannot foresee any other adverse effects.
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)?
Human genes will be sourced from the University of Otago stocks, which are covered by
GMD002066 approval. No human genetic material of Maori origin and no genetic
material of native plants or mammals will be used.
Are there any other potential adverse effects?
We cannot foresee any other ethical considerations.
No adverse effects have been identified.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Section Six – Additional information
Refer to page 31 of the user guide for assistance in completing this section
Additional Information
Y/N
Do any of the organism(s) need
approvals under any other New
Zealand legislation?
No
Does New Zealand have any
international obligations relating to
(any of) the organism(s)?
No
Have any of the new organism(s) in
this application previously been
considered in New Zealand or
elsewhere?
Yes
If yes, explain
This application seeks to extend the range
of host organisms for our current work,
which is covered by GMD08007.
M. smegmatis strains have previously been
approved for other institutions under
GMD07008
Is there any additional information
that you consider relevant to this
application that has not already been
included?
No
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?
No
The GMOs will be only used for the
expression of protein for the purpose of
purifying and characterising enzymes.
Animals or plants will not be exposed to
the GMOs.
Provide a glossary of scientific and technical terms used in the application:
Not applicable.
List of appendices attached:
Appendix 1: Examples of genes to be expressed in an E. coli or M. smegmatis host.
List of references attached:
Not applicable.
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Develop in containment a project of low risk genetically modified organisms by rapid assessment
Appendix 1: Examples of genes to be expressed in E. coli or M. smegmatis
Enzyme
Source organism(s)
Dihydrodipicolinate Escherichia coli, Mycobacterium tuberculosis,
synthase
Thermotoga maritima, Bacillus anthracis
Dihydrodipicolinate Escherichia coli, Mycobacterium tuberculosis,
reductase
Thermotoga maritima, Bacillus anthracis
Pyruvate kinase
Escherichia coli
N-acetyl
neuraminate lyase
Escherichia coli
TEV protease
Tobacco etch virus
Phosphotriesterase
Pseudomonas diminuta, Agrobacterium
radiobacter
Cytochrome P450
Bacillus megaterium
GroEL
Escherichia coli
GroES
Escherichia coli
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