GusPlus Overview

GUSPlus™ is a new reporter gene for use in molecular biology. There are GUSPlus vectors for checking transformations and screening transformants, and special vectors for use with TransBacter strains. The GUSPlus gene was originally isolated from Staphylococcus sp. but has been extensively altered by codon optimisation for expression in plants. It is described in Nature 433:629ff and in patents and patent applications including US patents 6,391,547, 6,641,996, 7,087,420 and 7,141,719. Issued in Australia as Patent Number 760275. Issued in New Zealand as Patent Number 1485. Published as WO 00/055333 and WO 99/13085 and pending in Brazil, Canada, Europe and Israel.

A pCAMBIA GUSPlus kit contains the following plasmids:

pCAMBIA1105.1, pCAMBIA1305.1, pCAMBIA1305.2, pCAMBIA0305.1, pCAMBIA0305.2, pCAMBIA1105.1R and pCAMBIA0105.1R

Detailed Description of GUSPlus

The beta-glucuronidase (GUS) enzyme from E. coli has been widely used to study promoter activity in different tissues or developmental stages. Recombinant DNA constructs are made in which the reporter gene (GUS) is attached to a promoter region of interest and the construct transformed into a cell or organism. However, it has some limitations that are overcome by a second generation of GUS gene developments at CAMBIA, which we are now making available in open source licenses. The GUSPlus gene was originally isolated from Staphylococcus sp. but has been extensively altered by codon optimisation for expression in plants.

Benefits of GUSPlus compared to E.coli GUS:

• Greater sensitivity, leading to faster colour development with X-glcA substrate
• Can be secreted, allowing non-destructive assay
• Higher thermal stability
• Greater tolerance to fixatives

CAMBIA has now developed vectors that include GUSPlus.

GUSPlus is a more sensitive reporter gene than E.coli GUS

GUSPlus can be used to screen for transformed tissue/plants

We have established efficient plant transformation protocols relying on screening transformed tissue using GUSPlus. Previous attempts to use a beta-glucuronidase for non-destructive assays were hampered by the need to use high concentrations or long incubations with X-glcA. The new GUSPlus gene overcomes these problems. Download the X-glcA assay protocol. (info)

• Rice and tobacco plant cells expressing secreted GUSPlus can be identified without compromising their regeneration capability.
• Arabidopsis seedlings expressing GUSPlus can be identified at the cotyledon stage and grown to mature plants.

Read more:

• Read about some of the new directions in research using these genes.
• Why is GUSPlus better? View a presentation on GUSPlus properties (info) that GUSPlus has compared to E.coli GUS
• Compare the five different GUSPlus vectors.
• View a presentation on the use of GusPlus as a selectable marker (info) for plant transformation.

The initial work on GUSPlus was carried out as a PhD project by Tuan Nyugen and a publication is expected soon. The work is being supported by the Rockefeller Foundation, Horticulture Australia and Monticello Research Australia.

Ordering Legacy pCAMBIA

 Please check CAMBIA materials website 

New Uses for Glucuronidase Genes and Proteins

Historically CAMBIA is strongly associated with the ß-glucuronidase gene (gusA) from E.coli. There are thousands of publications documenting its extensive use and versatility as a marker gene for plant genetic transformation and molecular physiology studies.

More recently the hydrolytic capabilities of the gusA gene have been exploited to release aglycones from glucuronides. This principle can be exploited widely either to increase phloem transportability of hydrophobic substances and/or to reactivate inert, biochemical activity compounds to development of novel, second-generation hydrolytic enzymes with improved characteristics (e.g. secretability into the apoplast of plant tissues, improved higher thermal and chemical stability to expand on existing histochemical assay conditions, variants in substrate specificity and processability).

A different approach consists in the development of transport mechanisms based on substrate-specific permeases, to trap the substrates in the cell. Phloem-translocatable, bioactive pro-compounds are being developed as target substrates for the activating enzymes.

Primer on Glucuronide Metabolism

Glucuronidation is one of the main detoxification pathways of xenobiotics but also self-metabolites in vertebrates. Microbial intestinal flora and other microorganisms have developed hydrolytic capabilities to obtain the glucuronate moiety as a carbon source.

In E. coli, an operon constituted of four genes, encoding a repressor, a glucuronidase, a permease, and a porin-like protein, is responsible for import and hydrolysis of glucuronide. Free glucuronate is then funnelled into metabolic pathways.

Substrates

The development of novel substrates for tissue-specific hydrolases, substrates having specific chemical, biochemical and physiological aspects, can contribute to selection and synthesis of agrichemical glucuronides, development of derivatives for positive selection, gametocidic or toxic chemicals.

Candidates have been selected based on their chemical suitability to provide derivatives. Another criterion is inactivity as a glucuronide and also non-toxicity to humans and non-target organisms. This also reduces the exposure of non-target pests to pesticides, reducing resistance buildup.

Another advantage of the approach is that the attachment of glucuronic acid makes many substances phloem translocatable, widening the scope of applicability for many agrichemicals whose use, although ecologically compatible, has been restricted by the lack of phloem mobility.

Positive Selection

Various projects at CAMBIA have attempted to develop screening and positive selection systems for plant transgenesis. This concept, first proposed by CAMBIA a number of years ago, would address some of the problems associated with currently used selectable marker genes:

• In certain jurisdictions, intellectual property rights restrict access to most practical negative selection methods.
• Public acceptance questions use of certain selectable marker genes, notably genes encoding resistance to antibiotics.
• Some regulatory agencies discourage use of such systems in plants to be released into the field.
• Selection systems based on genes conferring resistance to antibiotics or herbicides ("negative selection") also stress living transgenic cells, in part by causing the release of phenolic substances from non-transformed, dying plant cells that may affect growth of transformed cells. This effect, which can hurt regeneration, limits their value in challenging tissue culture systems.

The goal of CAMBIA's work in this area, not yet fully realised, has been to develop a positive-selection system based on a glucose-releasing procompound that circumvents these limitations. In plants, the disaccharide sucrose releases glucose when hydrolyzed by the enzyme invertase. The glucose released can then act an energy substrate to drive plant growth. In an attempt to "mimic" this principle, we chose the disaccharide cellobiouronic acid (CbA) as the substrate for our selection system. CbA is composed of glucuronic acid linked ß(1-4) to glucose. As CbA was not commercially available when this work began, CAMBIA had to develop a new method to prepare the sugar. US Patent US 6,268,493 describes this work and can be licensed royalty-free under open source principles for both research and commercial uses; potential commercial suppliers are of interest. Because of its structure, we expect CbA to be hydrolyzed by any of a variety of different ß-glucuronidase (GUS) enzymes. Plant cells lacking GUS activity are unable to grow on CbA as the sole carbon source. Transformation with a gene encoding GUS, however, would allow them to access the glucose "immobilized" within CbA and use it as an energy source for growth.

Additional advantages of the CbA/GUS selection system being developed are that CbA can be produced from gellan gum, a commonly used food additive in ice creams and other products, and that GUS has already been comprehensively tested for safety in products approved by many national regulatory agencies for human consumption. The ß-glucuronidase (GUS) enzyme that currently works best for this application was isolated from Thermotoga and together with the gene that encodes it is described in published patent applications WO 00/055333 and US 2003229921, and in U.S. Patent No. 7,087,420.

The license CAMBIA offers covers the gene and others similar to it, the protein, and a variety of uses of the protein, which may have industrial applications beyond positive selection. This enzyme is of interest for other applications because it is highly thermostable, and it can be licensed royalty-free under open source principles. We expect that continuing work by anyone who wishes to collaborate within the open source licensee community will optimize the enzyme characteristics for cleavage of CbA in the spaces around the growing cells. CAMBIA has done some codon manipulation toward getting it to work in plants, still not entirely successful.

For positive selection, due to dominating patents (see www.patentlens.net) there may not be freedom to operate in all jurisdictions even with the workable substrate and gene variants, but in many countries you would be free to research and even commercialise this concept, and we definitely welcome collaboration. We have been developing a technology landscape around positive selection, on which we also welcome collaboration.

Nomenclature of pCAMBIA GusPlus vectors

The numbering system works as follows:

• First digit - indicates plant selection: 0 for absence; 1 for hygromycin resistance; 2 for kanamycin.
• Second digit - indicates bacterial selection: 1 for spectinomycin/streptomycin resistance; 2 for chloramphenicol; 3 for kanamycin; 4 for spec/strep and kanamycin.
• Third digit - indicates polylinker used: 0 for pUC18 polylinker; 8 for pUC8 polylinker; 9 for pUC9 polylinker.
• Fourth digit - indicates reporter gene(s) present: 0 for no reporter gene; 1 for E.coli gusA; 2 for mgfp5; 3 for gusA:mgfp5 fusion; 4 for mgfp5:gusA fusion; 5 for Staphylococcus sp. gusA (GUSPlus).
• Fifth digit - the .1 denotes the absence of a signal peptide from the GUSPlus protein and the .2 denotes the presence of the GRP signal peptide for in planta secretion of the GUSPlus protein.

Quick-pick table (clicking on a plasmid number will navigate to the Genbank file)

GUSPlus™ Vector Comparison Chart

GUSPlus is now available in the following vector formats (see also BioForge Transbacter , for other or more specific vectors that are designed specifically for use with Transbacter):

pCAMBIA1305.1 (Genbank acc no. AF354045).

• This vector is based on pCAMBIA1301, in which the E. coli gusA gene has been replaced by GUSPlus. It is a compact binary vector with the pBR322 ori and bom sites for high copy replication in E.coli and transmission by mating (or electroporation). It contains the broad host range pVS1 ori for low copy, stable replication in Agrobacterium and other gene transfer competent bacteria. It contains the hygromycin resistance gene (hptII) for selection in plants. The GUSPlus gene contains an intron from the castor bean catalase gene to prevent expression by bacteria and ensure detection of plant-expressed glucuronidase activity. Researchers can excise the GUSPlus gene and insert their own gene of interest in its place or use these vectors to create fusions of GUSPlus with their gene of interest (If you have created a pCAMBIA vector derivative and would like to share it with other researchers, please let us know ).
• View 1305.1 Vector Map (info). (Download VNTI Viewer)

pCAMBIA1305.2 (Genbank acc no. AF354046).

• Same as pCAMBIA1305.1 except for the presence of the rice glycine-rich protein signal peptide sequence (GRP) inserted between the NcoI and BglII sites.
• View 1305.2 Vector Map (info). (Download VNTI Viewer)

pCAMBIA1105.1

• Same as pCAMBIA1305.1 except that the nptII bacterial kanamycin resistance gene has been replaced by aadA1, conferring resistance to spectinomycin and streptomycin. Derivative vectors such as 1105.1RC or 1105.1U have different multiple cloning sites to suit various needs.
• View 1105.1 Vector Map (info). (Download VNTI Viewer)

pCAMBIA1105.1R

• Same as pCAMBIA1105.1 except that the 322bp PvuII polylinker fragment has been replaced by a 403bp polylinker fragment from pCR2.1 (Invitrogen). At CAMBIA the R-vectors are used exclusively in non-Agrobacterium species. By following this practice we can be cautiously confident that transformants displaying the different polylinker sequence result from transformation another gene transfer competent bacterium and not from contaminating A.tumefaciens. We suggest that other users follow this practice wherever possible. pCAMBIA1105.1RRC has a modified multiple cloning site with rare enzymes to facilitate various cloning strategies.
• View 1105.1r Vector Map (info). (Download VNTI Viewer)

pCAMBIA0105.1R

• Derived from pCAMBIA1105.1 and contains the GRP-GUSPlus gene for secreted GUS activity. This and pCAMBIA 0305.2 are the vectors of choice for generating transformants without using a selectable marker gene. However, you are still using the 35S promoter (unless you replace it), which could be subject to the IP rights of others in some jurisdictions. We're trying to work around that too - if you have a replacement constitutive promoter you'd like to make widely available for use and improvement through a BiOS license, please talk to us!
• View 0105.1r Vector Map (info). (Download VNTI Viewer)

pCAMBIA0305.2

• Derived from pCAMBIA1305.2 and contains the GRP-GUSPlus gene for secreted GUS activity, but no selectable marker gene.
• View 0305.2 Vector Map (info). (Download VNTI Viewer)

• Do you want to order pCAMBIA GUSPlus vectors?
• Want more information?

See Frequently Asked Questions - GUSPlus Vectors
Read more about the original pCAMBIA vectors using the E. coli GUS gene on the CAMBIA website.

Full Technical Reports

An improved reporter system based on a novel β-glucuronidase (GUS) from Staphylococcus sp.

PhD thesis by Tuan Anh Nguyen.

Chapter 1 (info): General Introduction. (228.9 K)
Chapter 2 (info): Construction of the synthetic gusASsp gene and its variants. (199.3 K)
Chapter 3 (info): Purification of β-glucuronidases and preparation of their antibodies. (224.2 K)
Chapter 4 (info): Expression and secretion studies of GUSEco and GUSSsp in yeast. (113.9 K)
Chapter 5 (info): Expression and secretion studies of GUSEco and GUSSsp in plants. (142.1 K)
Chapter 6 & 7 (info): Investigation of some applications based on GUSSsp and Conclusion and prospects. (217.7 K)

Abstract

The GUS reporter system, based on the enzyme beta-glucuronidase (GUS) from E. coli, has been the most widely used tool in plant molecular biology in the last decade. This thesis reports various studies on a different GUS with superior biochemical characteristics, isolated from a soil Staphylococcus sp. bacteria.

Because of the high AT content of the native coding gene, a codon-optimized version of it was constructed and tested in E. coli, yeast and plants. The protein was expressed in E. coli and purified to high homogeneity for biochemical studies and antibody production.

In yeast, both E. coli GUS and Staphylococcus GUS, when provided with the invertase signal peptide, were efficiently secreted, and mostly localized in the periplasmic space. This is the first report of efficient secretion of E. coli GUS in yeast. All Staphylococcus GUS variants (wildtype, N118Q, and C499A mutants) were also secreted with high efficiency.

In plants, only Staphylococcus GUS was secreted when provided with either the GRP or extensin signal peptide. E. coli GUS was not secreted, consistent with earlier reports. The use of an intron-containng version of the synthetic Staphylococcus gusA as an improved reporter in plants was validated. Staphylococcus GUS was shown to offer faster and more sensitive histochemical detection. Its activity was also better detected after tissue fixation, allowing more precise histochemical localization of the enzyme. With secreted Staphylococcus GUS, non-destructive GUS assays were possible with tissues such as calli and roots. Tissue specific manipulation system using secreted Staphylococcus GUS was also demonstrated, with the example of the secreted Staphylococcus GUS driven by a pollen-specific promoter ntp303.

Frequently Asked Questions - Obtaining and Using pCAMBIA GUSPlus Vectors

• Why should I use GUSPlus?
• What are pCAMBIA vectors?
• How can I obtain GUSPlus pCAMBIA vectors?
• What version of GUSPlus is used in pCAMBIA vectors?
• Do pCAMBIA GUSPlus vectors have a hexa-His tag appended to the reporter gene?
• What concentration of bacterial antibiotic should I use?
• What concentration of plant selection antibiotic should I use?
• What about the original pCAMBIA vectors?
• My friend works in a lab that has GUSPlus pCAMBIA vectors. Can I ask for the vectors? Do I still need to pay?
• Will I have full freedom to use pCAMBIA vectors?
• What are the BiOS License conditions relevant to GUSPlus?
• Will I have to pay royalties to use GUSPlus?
• Is GUSPlus patented or owned by any for-profit company?
• What is the Staining Protocol for GUSPlus?

See GUSPlus Vector Comparison Chart for a description of the GUSPlus Vectors available from CAMBIA.

Why should I use GUSPlus?

The GUSPlus protein has better catalytic and chemical resistance properties than Escherichia coli GUSA. GUSPlus also has the great advantage in that it can be secreted by plant cells (see GUSPlus Detailed Description for more information). Also, GUSPlus is being offered widely to both non-profit and for-profit organizations that are willing to agree to the terms of a BiOS license. Under this license, the genes may be used in research or for commercial product development royalty-free and without payment of additional license fees, but only under the condition that all improvements are granted back to the common group of licensees. This license structure, pioneered within the BiOS Initiative, is to ensure that a choice by any licensed innovator to use these genes has the best chance to lead to deliverables, as opposed to becoming encumbered in patent thickets that prevent productive use.

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What are pCAMBIA vectors?

CAMBIA has a small team that made these vectors to facilitate research and we've continued to make them widely available below cost for this reason. See details about the pCAMBIA vector backbone and how it was constructed at Description of the pCAMBIA vectors.

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How can I obtain GUSPlus pCAMBIA vectors?

We provide GUSPlus pCAMBIA vectors for research by non-profit organisations, or for profit companies. To obtain GUSPlus pCAMBIA vectors for research by non-profit organisations, simply complete the GUSPlus Request Form (info) and send it to us by mail or fax. To request a license for use within a company or commercial environment, contact us at licenses@cambia.org. Either way the request comes to us, we will then send you a request confirmation e-mail with a tracking number. You will be responsible to get any payments due to us to help us cover our costs in sending these vectors to you.

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What version of GUSPlus is used is used in pCAMBIA vectors?

pCAMBIA1105.1, 1105.1R, 1305.1 and 1305.2 use a fully synthetic, codon and expression-optimized version of a glucuronidase-encoding gene from a Staphylococcus species isolated by CAMBIA. These plasmids are available under CAMBIA's BiOS license. In pCAMBIA1305.2 the GusPlus gene is fused to a sequence encoding the glycine rich protein secretion signal peptide. The secretion signal results in the GUSPlus protein being efficiently transported to the periplasmic (or apoplastic) space, where it is active. This allows non-destructive in vivo staining and detection in living tissues that can be grown further after staining. A castor bean catalase gene intron inserted near the 5' end of the GUSPlus gene eliminates expression of GUSPlus in bacteria, but allows expression upon transfer to a plant cell. This make monitoring of gene expression associated with transformed tissues more informative and less liable to artifactual intepretation. The presence of the intron also improves the overall expression levels of GUSPlus protein in some cases, probably by stabilizing the mRNA.

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Do pCAMBIA GUSPlus vectors have a hexa-His tag appended to the reporter gene?

Yes. In each vector the hexa-His tag is at the C-terminus of the open reading frame.

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What concentration of bacterial antibiotic should I use?

Both pCAMBIA1305.1 and 1305.2 confer kanamycin (aphIII/nptIII/3'5''-aminoglycoside phosphotransferase typeIII from Enterococcus faecalis pJH1) resistance. For selection of E. coli and Agrobacterium tumefaciens cultures use 50 µg of kanamycin per mL of media.

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What concentration of plant selection antibiotic should I use?

pCAMBIA1305.1 and 1305.2 both contain the hph resistance gene, obtained from a Klebsiella species via E. coli and the conferred transformed plant resistance is to Hygromycin B. For selection in Arabidopsis use 50 µg/mL in germination medium. For rice, we suggest you use 50 µg/mL in 2N6 and then in RGH6, and for tobacco we suggest 50 µg/mL in RMOP and similarly in MST. You may, however, find that no selection is needed. In our hands GUSPlus works well for screening transformants because it can be assayed non-destructively.

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Will I have full freedom to use pCAMBIA vectors?

Without knowledge of the detail of what you are trying to do it is not possible to make any assertions about this, and CAMBIA cannot warrant it. Many aspects of what you may want to do, for example the use of Agrobacterium to transform plants, and some DNA sequences within the pCAMBIA vectors, such as the 35S promoters and the hygromycin resistance gene, are covered by patents issued to other entities in many countries. Although you may have a research exemption (either de factode jure) or certain aspects of your work may not be covered by a patent in your country, it could be subject to patents where products developed from your work might later be used or exported. In such circumstances the owner(s) of the patent rights might legally prevent deliverability of the results of your research work. This is one of the most important reasons for the BiOS license developed by CAMBIA, and available under the same license CAMBIA has developed and is improving a plant transformation system that will be less limited by restrictive licensing of patented technologies. If you want to learn more about this project, or join in the community of people trying to make it happen, please visit the TransBacter project.

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What about the original pCAMBIA vectors?

If you are working on a non-commercial plant research project in a public institution, you can still request all the other pCAMBIA vectors from CAMBIA. Details and requst form can be found on the CAMBIA website.

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My friend works in a lab that has pCAMBIA GUSPlus vectors. Can I ask for the vectors? Do I still need to pay?

Please fill out and sign the GUSPlus Request Form (info) and send it to us. Once you have registered, it is possible to get vectors from your colleague. Please note on the form who will supply you with the vectors, and cross out the payment section (as the charges are solely to cover production and distribution, payment is not required if obtaining strains from a friend or colleague).

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What are the BiOS License conditions relevant to GUSPlus?

In summary, in return for CAMBIA's permission to use the technologies, a licensee institution agrees to allow and encourage its employees and students to post any improvements made to the technologies on this website. The licensee institution also agrees not to assert any intellectual property rights to the improvements against other licensees. All licensees that comply with these license terms are allowed to use the technology for research, public good, and/or commercial product development. Companies with for-profit governance also agree to assist us in meeting costs such as providing this website and any improved protocols that are developed. However, CAMBIA's BiOS license does not require any commitment to repay patenting costs, nor any milestone payments or royalties.

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Will I have to pay royalties to use GUSPlus?

BiOS licenses are available from CAMBIA completely royalty-free for research and commercial purposes. For-profit companies based in OECD countries such as Australia and the USA are normally requested to enter into a technology subscription agreement, or a similar arrangement that helps CAMBIA defray costs of keeping the technology available on the BioForge. For-profit companies based outside the OECD can take up such a subscription for a reduced fee or in-kind assistance (please contact us for information), and non-profits obtain all the benefits of the license and technology support subscription without paying a fee. CAMBIA has committed to this broadly accessible licensing arrangement in the hope that many who wish to see the protected commons of improvements expanded for public good will use this technology.

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Is GUSPlus patented or owned by any for-profit company?

The patents issued in the USA, Australia and New Zealand, and patent applications still pending in multiple countries, are owned by CAMBIA, which is a non-profit registered in Australia and a non-government organisation registered with the Food and Agriculture Organization. CAMBIA does not receive any profit from selling, using, or out-licensing GUSPlus. However, the technology has been patented at CAMBIA's expense in order to manage the rights to practice the technology in accordance with the principles of "open source". Any entity may obtain a license to use the technology only by agreeing to grant back improvements to all other licensees. The same license allows the use of CAMBIA's Transbacter technology (see Transbacter project on this website).

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What is the Staining Protocol for GUSPlus?

Standard GUSPlus/PenGUS staining protocol

modified from Jefferson RA (1987) Plant Mol. Biol. Rep. 5:387-405)
Standard X-GlcA solution
50mM sodium phosphate buffer pH7.0
10mM EDTA pH8.0
0.1% (v/v) Triton X-100
0.5mg/mL X-GlcA
Store at 4°C in dark

Cover tissue with standard X-glcA solution, vacuum infiltrate for 5 mins. Incubate at 37°C for up to 16h to develop blue colour of product.

Non-destructive GUSPlus/PenGUS staining protocol--more recently developed at CAMBIA

This method has been tested with GUSPlus and PenGUS (links to the relevant BioForge pages) on rice callus, tobacco shoots and Arabidopsis seedlings and does not prevent continued growth of these tissues. Note that we do not warrant that it may not be detrimental to other tissues or to individual cells. There are a number of modifications/additions to the standard GUS protocol that can be found in the literature, depending on your tissue, and you can experiment with toxicity.

Cover tissue with 200 ug/mL X-glcA in 20mM sodium phosphate buffer pH7.0. This is a standard buffer without Triton X-100 or any other detergent or surfactant. Do not use vacuum infiltration or a 37°C incubation, as this will have a negative effect on cell survival and growth. Incubate at room temperature until blue colour appears.

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New Eukaryotic GUS Gene - "FunGus"

A fungal beta-glucuronidase has been discovered, described in the patent publications US 2005/153448 and WO 2005/68617 A2, and in a recent publication. Patent claims have recently issued in the United States in US Patent 7,148,407. This gene has closer codon usage to that of other eukaryotes than that of bacterial glucuronidase genes.

We have successfully used this gene in reporter constructs, pCAMBIA0306.2 and pCAMBIA1306.2 (see VectorNTI diagrams attached--download using VNTI Viewer), for screening transformation in rice.

Patents are about to issue on this gene. Accordingly, these constructs will become available from CAMBIA in early 2008 under the same terms as the GUSPlus constructs described on other BioForge pages, i.e. under the BiOS License.

Frequently Asked Questions

• Why should I use FunGUS?
• How can I obtain pCAMBIA vectors containing FunGUS?
• Will I have full freedom to use pCAMBIA vectors?
• What are the BiOS License conditions relevant to FunGUS?
• Will I have to pay royalties to use FunGUS?

See page describing the nomenclature of pCAMBIA vectors available from CAMBIA.

Why should I use FunGUS?

The FunGUS protein is expressed naturally in a eucaryote, unlike Escherichia coli GUSA. Like GUSPlus, it also has the great advantage in that it can be secreted by plant cells (the gene is fused to a sequence encoding the glycine rich protein secretion signal peptide which results in the GUSPlus protein being efficiently transported to the periplasmic or apoplastic space, where it is active). This allows non-destructive in vivo staining and detection in living tissues that can be grown further after staining. Also, FunGUS is being offered widely to both non-profit and for-profit organizations that are willing to agree to the terms of a BiOS license. Under this license, the genes may be used in research or for commercial product development royalty-free and without payment of additional license fees, but only under the condition that all improvements are granted back to the common group of licensees. This license structure, pioneered within the BiOS Initiative, is to ensure that a choice by any licensed innovator to use these genes has the best chance to lead to deliverables, as opposed to becoming encumbered in patent thickets that prevent productive use.

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How can I obtain pCAMBIA vectors containing FunGUS?

pCAMBIA FunGUS Vectors (pCAMBIA0306.2 and 1306.2) are currently unavailable but we aim to provide as soon as we have the resources available. These plasmids will be available under CAMBIA's BiOS license or a BiOS-compliant MTA. Contact us at materials@cambia.org.

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Will I have full freedom to use pCAMBIA vectors?

Without knowledge of the detail of what you are trying to do it is not possible to make any assertions about this, and CAMBIA cannot warrant it. Many aspects of what you may want to do, for example the use of Agrobacterium to transform plants, and some DNA sequences within the pCAMBIA vectors, may be covered by patents issued to other entities in many countries. Although you may have a research exemption (either de facto or de jure) or certain aspects of your work may not be covered by a patent in your country, it could be subject to patents where products developed from your work might later be used or exported. In such circumstances the owner(s) of the patent rights might legally prevent deliverability of the results of your research work. This is one of the most important reasons for the BiOS license developed by CAMBIA, and available under the same license CAMBIA has developed and is improving a plant transformation system that will be less limited by restrictive licensing of patented technologies. If you want to learn more about this project, or join in the community of people trying to make it happen, please visit the TransBacter project. pCAMBIA0306.2 lacks the hygromycin resistance gene covered by Syngenta patents in some jurisdictions, and was designed for use in screening for transformants non-destructively without antibiotic selection. We have successfully used it in this way for obtaining transformants in both high-frequency and low-frequency plant transformation systems, notably for rice transformation.

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What are the BiOS License conditions relevant to FunGUS?

In summary, in return for CAMBIA's permission to use the technologies, a licensee institution agrees to allow and encourage its employees and students to post any improvements made to the technologies on this website. The licensee institution also agrees not to assert any intellectual property rights to the improvements against other licensees. All licensees that comply with these license terms are allowed to use the technology for research, public good, and/or commercial product development. Companies with for-profit governance also agree to assist us in meeting costs such as providing this website and any improved protocols that are developed. However, CAMBIA's BiOS license does not require any commitment to repay patenting costs, nor any milestone payments or royalties.

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Will I have to pay royalties to use FunGUS?

BiOS licenses are available from CAMBIA completely royalty-free for research and commercial purposes. For-profit companies based in OECD countries such as Australia and the USA are normally requested to enter into a technology subscription agreement, or a similar arrangement that helps CAMBIA defray costs of keeping the technology available on the BioForge. For-profit companies based outside the OECD can take up such a subscription for a reduced fee or in-kind assistance (please contact us for information), and non-profits obtain all the benefits of the license and technology support subscription without paying a fee. CAMBIA has committed to this broadly accessible licensing arrangement in the hope that many who wish to see the protected commons of improvements expanded for public good will use this technology. The patents covering FunGUS are owned by CAMBIA, which is a non-profit registered in Australia and a non-government organisation registered with the Food and Agriculture Organization. CAMBIA does not receive any profit from selling, using, or out-licensing FunGUS. The technology has been patented at CAMBIA's expense in order to manage the rights to practice the technology in accordance with the principles of "open source". Any entity may obtain a license to use the technology only by agreeing to grant back improvements to all other licensees. The same license allows the use of CAMBIA's GUSPlus, transactivation and Transbacter technologies (see other projects on this BioForge website).

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The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.