Cambia is an independent non-profit institute creating new technologies, tools and paradigms to promote change and enable innovation. Information on pCAMBIA may be found belowas well as in the BioForge Transbacter Project. While Cambia is no longer distributing these vectors, you can still request them from any other lab that uses or publishes using them they were available under the open source terms of a BiOS license info. Plant transformation is now routine in hundreds of laboratories worldwide, using bacterially-mediated or direct DNA transfer methods such as bombardment.

These methods have both technical and intellectual property limitations see the BioForge TransBacter Projectin which we're improving the alternative to Agrobacterium transformation for more complete freedom to operate. One of the biggest technical limitations laboratories face is that many vectors still used are historical relics with substandard features that make DNA constructions awkward or cumbersome:.

While not perfect and having technical and IP limitations see the BioForge GUSPlus Projectin which we're improving reporter genes and selection methods for more complete freedom to operatepCAMBIA vectors offer:. The pUC18 polylinker was used in some vectors, but pUC8 and pUC9 polylinkers were also used to simplify the choice of cloning enzyme.

In the age of PCR, it is no longer necessary to have a large number of cloning sites. The smaller polylinkers also eliminate potential conflicts from sites such as Sph I which has an ATG or Xba I which has a TAG.

binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid

This makes other sites in the vector more useful such as the Sph I site outside the right T-DNA Border, or the Sac II site outside the left T-DNA Border. Plant selection genes in the pCAMBIA vectors are driven by a double-enhancer version of the CaMV35S promoter and terminated by the CaMV35S polyA signal. NOTE that this 35S promoter can have an enhancer effect on the expression of other genes in the same cassette, so gene expression results using pCAMBIA derivatives in which portions of this promoter are still present should be interpreted with caution.

Furthermore, it is your responsibility to check whether the 35S promoter or any other components you use are subject to patents in your country. You can find help with this at CAMBIA's Patent Lens website.

Reporter genes feature a hexa-Histidine tag at the C-terminus to enable simple purification on immobilised metal affinity chromatography resins. The sequence for this tag occurs between the first Nhe I site there is a second Nhe I site in the pVS1-rep that we didn't eliminate and the unique Pml I site. Genes of interest may be inserted in place of the reporter gene. Insertion without a stop codon and in frame at the first Nhe I site will append a hexa-Histidine tag to your protein of interest.

Insertion without a stop codon and in frame at the Pml I site will append a stop codon. Insertion at the Bst EII site will add neither a tag nor a stop codon so you may want to ensure that a sequence inserted here contains a stop codon. First digit - indicates plant selection: Second digit - indicates bacterial selection: Third digit - indicates polylinker used: Fourth digit - indicates reporter gene s present: Fifth digit - notes some other special feature.

So far this has been used only with: Due to resource limitations, not all possible vector feature combinations have been created at CAMBIA. You may initially be disappointed to find that we don't have, for example, a pCAMBIA The vectors were designed however, such that it should be a relatively simple matter for a researcher needing such a vector to construct it from the components in other vectors.

If you have created a pCAMBIA vector derivative that other researchers will find useful and you want to share with other researchers, email us. This vector is a part of a new series of pCAMBIA GT-BACK vectors gene Transfer-Bacterial Acquired Competence with kanamycin selection that encompasses a modified version of pCAMBIA This new unitary vector allows the DNA transfer capability to be moved into, and stabilized in, a much wider range of bacteria and it will be provided as an open source toolkit.

The added-value features of the new vector over the Transbacter Technology are: This is the means by which literally thousands of laboratories worldwide have obtained and further sent out pCAMBIA vector sets.

If you wish to discuss or query any CAMBIA materials please login to the. These vectors contain a range of restriction sites on either side of the pUC8 or pUC9 polylinker, making them suitable for advanced construction purposes with users inserting their own promoters, selection genes, reporter genes, etc.

The only functional signals between the T-DNA borders are the start and stop codons, the histidine tag, and the NOS-poly A signal. All the standard features of the pCAMBIA backbone are present: Researchers wanting the latest versions should see pCAMBIA Information on the older pCAMBIA vectors is here for convenience.

These vectors contain minimal heterologous sequences for plant transformation and selection of transformants; they allow insertion of desired genes for transformation into plants but require all promoter and terminator sequences for plant expression of newly cloned genes.

The minimal selection vectors have one of two plant selection genes: In both cases the selection gene is driven by a double-enhancer version of the CaMV35S promoter.

These genes have been subjected to site-directed mutagenesis to eliminate interfering restriction sites within the coding sequence by silent changes. Two different bacterial resistance markers are provided kanamycin or chloramphenicolallowing a broad range of Agrobacterium or E. The full modular format is provided for convenient PCR cloning and how to get money to buy a ps3 expression.

For researchers performing promoter analysis the use of the minimal vector containing GUSPlus pCAMBIA Co-transformation strategies are desirable to physically separate in the transformed plant genome the promoter of the plant selection gene usually 35S and the promoter of interest often much weaker or more specific driving a gus or other reporter gene.

The way that we have been doing this for years is that two vectors are separately transformed into the same strain of Agrobacterium or more preferably strains from the BioForge TransBacter Projectfor freedom to operatebut obviously co-transformation can also be done by simultaneously transforming with two separate isolates each containing one of the vectors.

If using one isolate, single colonies are selected, the plasmid DNA analysed, the cells induced on special media if required for your plant speciesand the two cell lines are mixed together immediately prior to application onto the plant tissues to be transformed. These vectors contain a fully functional gus A reporter construct for simple and sensitive analysis of gene a binary plant vector strategy based on separation of vir- and t-region of the agrobacterium or presence in regenerated plants by GUS assay.

The construct uses E. The gusA reporter gene is cloned in new modular format. These plasmids are suitable for insertion of other genes of interest containing their own promoter and terminator.

Metatrader forex can excise the gusA gene and insert their own gene of interest in its place or use these vectors to create fusions of gusA with their gene of interest if you have created a pCAMBIA vector derivative that other researchers will find useful and would like to share it, please let us know. These vectors contain the pUC18 polylinker- lacZa and the same bacterial and plant selection genes as their corresponding Minimal Selection Vectors.

For those desiring the best of both worlds in reporter genes we constructed these vectors, similar in utility to the GUS Intron Selection Vectors GIS mcx commodity market tips, but including GFP.

Being a non-catalytic protein places an intrinsic limit on detection sensitivity with fluorescent proteins and expensive equipment is needed for quantitative assays and microscopic observation. GFP is nonetheless popular as a reporter gene and we provide it cloned in full New Modular format for those wishing to use it. These vectors are based on pCAMBIA bacterial kanamycin resistance, plant hygromycin selection, pUC18 polylinker in lacZa but contain the mgfp 5 version of the Aequoria victoria green fluorescent protein Siemering et al.

These are intronless versions of gusA NQso there is the possibility that expression in primary transformants is the result of expression of the reporter proteins by residual Agrobacterium tumefaciens cells or other bacteria present in plant cultures. Analysis of large numbers of transformants of rice and Arabidopsis at CAMBIA showed that the fluorescence produced by the MGFP5 protein was quite faint.

As a result of this our researchers constructed similar constructs using the egfp gene available from Clontech. Results with these proteins were far superior and, although we are unable to distribute vectors containing this gene, we recommend that researchers purchase pEGFP from Clontech and use this to construct their own plasmids analogous to pCAMBIA, pCAMBIA or pCAMBIA Designed to utilize gus A as a true reporter of gene expression by fusion construction, these vectors are derived from pCAMBIA andand contain a promoterless, non-intron gus A NQ gene without an initiation codon in three reading frames, and with either pUC8 or pUC9 oriented polylinkers.

This permits simple construction of carboxy-terminus protein fusions to gusA. Plant selection is with hygromycin, and bacterial selection with kanamycin.

DNA-Delivery Methods to Produce Transgenic Plants

The pCAMBIA and vectors may also be used for construction of transcriptional karachi stock exchange dividends translational fusions to gus A. They are similar to the Xa, b, c series though they retain the initiation codon of the Nco I site in the New Modular structure around the gus A NQ gene, and are only available in one reading frame.

Designed for promoter testing in plantathese vectors feature a promoterless version mylikes.com learn how to make money online gus A NQ with the catalase intron immediately downstream of a truncated lacZa containing either the pUC8 or pUC9 polylinker.

All plasmids in this series have hygromycin as the plant selection gene, and bacterial selection is available with either rahsia forex download pdf Z, Z or kanamycin Z, Z. Experience with these vectors has shown that the very strong 35S promoter in fact a a binary plant vector strategy based on separation of vir- and t-region of the agrobacterium version of it which drives the hptII gene in the T-DNA of these and most other pCAMBIAs causes significant interference in the expression patterns observed.

Interpret your results with caution! Idea inventionsuccess.net make marketing money control transformants created using one of these vectors without a promoter added upstream of the gusA gene show low to moderate level GUS expression in a range of tissues.

Enhancer-trap make money fast in montreal performed at CAMBIA using somewhat rearranged versions of these vectors have also shown consistent interfering expression which we attribute to the nearby 35S promoter. This artefactual expression may be exacerbated in experiments where researchers attempt to analyse trimmed-down versions of their promoters of interest lacking the natural insulating sequences of full-length promoters.

To avoid this 35S-interference from promoter analyses, we recommend using co-transformation strategies as described above in the section on the Minimal Selection Vectors. Such a vector might be called pCAMBIAZ, but we have never constructed it for distribution as part of the pCAMBIA vector kit. Agrobacterium use is constrained in jurisdictions such as the USA, and it may be unwise to use it in any research that might result in a product for sale or import into the USA.

You may wish to use the Transbacter system instead for freedom to operate. For information, see the BioForge TransBacter Project. Chen L, Zhang S, Beachy RN, Fauquet CM A protocol for consistent, large-scale production of fertile transgenic rice plants. Plant Cell Reports Christou P Production of transgenic rice Oryza sativa L.

Deblaere R, Reynaerts A, Hofte H, Hernalsteens JP, Leemans J, and Van Montagu M Vectors for cloning in plant cells. Hajdukiewicz,P, Svab, Z, Maliga, P. Plant Mol Biol Hiei Y, Ohta S, Komari T, Kumashiro T Efficient transformation of rice Oryza sativa L. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA Binary vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Hood EE, Helmer GL, Fraley RT, Chilton MD The hypervirulence of Agrobacterium tumefaciens A is encoded in a region of pTiBo outside of T-DNA.

Hood EE, Gelvin SB, Melchers S, Hoekema A New Agrobacterium helper plasmids for gene transfer to plants EHA Jefferson RA, Kavanagh TA, Bevan MW GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. Klapwijk PM, van Breukelen J, Korevaar K, Ooms G, Schilperoort RA T ransposition of Tn encoding streptomycin resistance into octopine Ti plasmid of Agrobacterium tumefaciens.

Lazo GR, Stein PA, Ludwig RA A DNA transformation-competent Ara bidopsis genomic library in Agrobacterium. Ohta S, Mita S, Hattori T, Nakamura K Construction and expression in tobacco of a beta-glucuronidase GUS reporter gene containing an intron within the coding sequence.

Plant Cell Physiol Ooms G, Hooykaas PJJ, Van Veen RJM, Van Beelen P, Regensburg-Tunk JG, Schilperoort RA Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region.

pCambia Vectors - Cambia - Enabling Innovation

Peralta EG, Hellmiss R, Ream W Overdrive, a T-DNA transmission enhancer on the A. Immobilized metal ion affinity chromatography. Protein Expre Purif 3: Siemering KR, Golbik R, Sever R, Haseloff J Mutations that suppress the thermosensitivity of green fluorescent protein.

Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron.

Nucl Acids Res While Cambia is no longer distributing these vectors, you can still request them from any other lab that uses or publishes using them they were available under the open source terms of a BiOS license info Much of the information below relates to older pCAMBIA vectors Description of the pCAMBIA vectors Quick-pick table and Genbank links Description of pCAMBIA vectors pCAMBIA Information on GUSPlus and penGUS, and vectors containing them Do-It-Yourself vectors Minimal Selection Vectors see special note for researchers wanting to test promoters Gus Intron Selection vectors GFP Selection vectors Fuse and Use vectors Promoter Cloner vectors Genotypes of some useful Agrobacterium tumefaciens strains Resources References Other Resources Description of the pCAMBIA vectors Plant transformation is now routine in hundreds of laboratories worldwide, using bacterially-mediated or direct DNA transfer methods such as bombardment.

One of the biggest technical limitations laboratories face is that many vectors still used are historical relics with substandard features that make DNA constructions awkward or cumbersome: While not perfect and having technical and IP limitations see the BioForge GUSPlus Projectin which we're improving reporter genes and selection methods for more complete freedom to operatepCAMBIA vectors offer: A few points about the pCAMBIA cloning strategy: Nomenclature of pCAMBIA vectors: The four digit numbering system works as follows: Minimal Selection Vectors pCAMBIA; pCAMBIA; pCAMBIA; pCAMBIA; pCAMBIA; pCAMBIA Researchers wanting the latest versions should see pCAMBIA Gus Intron Selection vectors pCAMBIA; pCAMBIA; pCAMBIA; pCAMBIA Researchers wanting the latest versions should see pCAMBIA GFP Selection vectors pCAMBIA; pCAMBIA; pCAMBIA For those desiring the best of both worlds in reporter genes we constructed these vectors, similar in utility to the GUS Intron Selection Vectors GISbut including GFP.

Fuse and Use vectors pCAMBIA and and their Xa, b, c ORF variants Designed to utilize gus A as a true reporter of gene expression by fusion construction, these vectors are derived from pCAMBIA andand contain a promoterless, non-intron gus A NQ gene without an initiation codon in three reading frames, and with either pUC8 or pUC9 oriented polylinkers.

Genotypes of some useful Agrobacterium tumefaciens strains Warning!! EHA, genotype C58 pTiBo; T-region:: EHA is a Km S derivative of EHA Hood et al. AGL1, genotype is AGL0 C58 pTiBo recA:: A, reconstructed strain, derivative of A cured C58 harboring pTiBo, super-virulent Hood et al. Selected References Chen L, Zhang S, Beachy RN, Fauquet CM A protocol for consistent, large-scale production of fertile transgenic rice plants. Cambia's projects and initiatives. Footer information This website is a service of Cambia.

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