Frequently Asked Questions
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). Under the BiOS license, the genes may be used in research or for commercial product development royalty-free and without payment of additional license fees.
What are pCAMBIA vectors?
Cambia is no longer distributing these vectors. As Cambia’s technology is now in the public domain, you can request the vectors from any other lab that uses or publishes using these vectors. Cambia is agreeable to these terms and does not require any payment or license..
How can I obtain GUSPlus pCAMBIA vectors?
Cambia is no longer distributing these vectors. As Cambia’s technology is now in the public domain, you can request the vectors from any other lab that uses or publishes using these vectors. Cambia is agreeable to these terms and does not require any payment or license.
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. 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 get the other pCAMBIA vectors from other labs. .
My friend works in a lab that has pCAMBIA GUSPlus vectors. Can I ask for the vectors? Do I still need to pay?
Yes, it is possible to get vectors from your colleague. Payment is not required if obtaining strains from a friend or colleague).
What are the BiOS License conditions relevant to GUSPlus?
In summary, CAMBIA’s technology is now in the public domain.
Will I have to pay royalties to use GUSPlus?
BiOS licenses are available from CAMBIA completely royalty-free for research and commercial purposes. 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?
CAMBIA decided to put its technology in the public domain . CAMBIA did not receive any profit from selling, using, or out-licensing GUSPlus. However, the technology was 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
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.