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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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).
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 darkCover 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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