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List of Legacy pCAMBIA Vectors

The complete kit of pCAMBIA legacy vectors includes the following plasmids:

pCAMBIA0380, pCAMBIA0390, pCAMBIA1200, pCAMBIA1201, pCAMBIA1281Z, pCAMBIA1291Z, pCAMBIA1300, pCAMBIA1301, pCAMBIA1302, pCAMBIA1303, pCAMBIA1304, pCAMBIA1380, pCAMBIA1381Xa, pCAMBIA1381Xb, pCAMBIA1381Xc, pCAMBIA1381Z, pCAMBIA1390, pCAMBIA1391, pCAMBIA1391Xa, pCAMBIA1391Xb, pCAMBIA1391Xc, pCAMBIA1391Z, pCAMBIA2200, pCAMBIA2201, pCAMBIA2300, and pCAMBIA2301

These vectors are discussed in more detail below:

pCAMBIA0380; pCAMBIA0390

These vectors contain a range of restriction sites on either side of the pUC8 (0380) or pUC9 (0390) 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: kanamycin bacterial selection, high copy number in E. coli, and stable replication in A. tumefaciens.

Minimal Selection Vectors

pCAMBIA1200; pCAMBIA1300; pCAMBIA1380; pCAMBIA1390; pCAMBIA2200; pCAMBIA2300

Researchers wanting the latest versions should see pCAMBIA 1305.1, pCAMBIA1105.1 or pCAMBIA 1305.2, which can be requested through the GUSPlus Project. 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: hptII encoding resistance to hygromycin, or nptII encoding resistance to kanamycin. 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 chloramphenicol), allowing a broad range ofAgrobacterium or E. coli strains to be used. The pUC18 polylinker within the lacZa fragment allows blue/white screening of clones in E. coli cloning work.

pCAMBIA1380 and 1390 are based on pCAMBIA1300, but the pUC18 polylinker-lacZa fragment has been deleted and replaced with the simpler pUC8 (1380) and pUC9 (1390) polylinkers, which do not contain potentially confounding start or stop codons. The full modular format is provided for convenient PCR cloning and gene expression.

For researchers performing promoter analysis the use of the minimal vector containing GUSPlus (pCAMBIA 0305.2, which can be ordered through the GUSPlus Project) is recommended, rather than any of the other pCAMBIA vectors. 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 TransBacter Project, for freedom to operate), but 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 species), and the two cell lines are mixed together immediately prior to application onto the plant tissues to be transformed. In our hands this method gives 10-30% of transformed plant lines containing both T-DNAs.

Gus Intron Selection vectors

CAMBIA1201; pCAMBIA1301; pCAMBIA2201; pCAMBIA2301

Researchers wanting the latest versions should see pCAMBIA 1305.1, pCAMBIA1105.1 or pCAMBIA 1305.2, which can be requested through the GUSPlus Project. Information on the older pCAMBIA vectors is here for convenience.

These vectors contain a fully functional gusA reporter construct for simple and sensitive analysis of gene function or presence in regenerated plants by GUS assay. The construct uses E.coli gusA (N358Q — to avoid N-linked glycosylation) with an intron (from the castor bean catalase gene) inside the coding sequence to ensure that expression of glucuronidase activity is derived from eukaryotic cells, not from expression by residual A.tumefaciens cells. 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. Researchers 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, 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.

GFP Selection vectors

pCAMBIA1302; pCAMBIA1303; pCAMBIA1304

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), 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 pCAMBIA1301 (bacterial kanamycin resistance, plant hygromycin selection, pUC18 polylinker in lacZa) but contain the mgfp5 version of the Aequoria victoria green fluorescent protein (Siemering et al., 1996) either alone – pCAMBIA1302 – or in translational fusion with gusA (N358Q) in both arrangements: pCAMBIA1303 has a gusA-mgfp5-His6 fusion, and pCAMBIA1304 has a mgfp5-gusA-His6 fusion. These are intronless versions of gusA (N358Q), so 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 pCAMBIA1302, pCAMBIA1303 or pCAMBIA1304. If you have created a pCAMBIA vector derivative that other researchers will find useful, please let us know .

Fuse and Use vectors

pCAMBIA1381 and 1391 and their Xa, b, c ORF variants

Designed to utilize gusA as a true reporter of gene expression by fusion construction, these vectors are derived from pCAMBIA1380 and 1390, and contain a promoterless, non-intron gusA (N358Q) 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.

The pCAMBIA1381 and 1391 vectors may also be used for construction of transcriptional or translational fusions to gusA. They are similar to the Xa, b, c series though they retain the initiation codon of the NcoI site in the New Modular structure around the gusA (N358Q) gene, and are only available in one reading frame.

If you have created a pCAMBIA vector derivative that other researchers will find useful, please let us know.

Promoter Cloner vectors™

pCAMBIA1281Z; pCAMBIA1291Z; pCAMBIA1381Z; pCAMBIA1391Z

Designed for promoter testing in planta, these vectors feature a promoterless version of gusA (N358Q) with the catalase intron immediately downstream of a truncated lacZacontaining 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 chloramphenicol (1281Z, 1291Z) or kanamycin (1381Z, 1391Z). The truncated lacZa is functional for blue/white screening of clones in suitable E.coli host strains.

Experience with these vectors has shown that the very strong 35S promoter (in fact a double-enhancer 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! Negative 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 experiments 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.

In such a strategy the promoter of interest can be cloned into one of our Promoter Cloner Vectors, but this should first be modified by performing a SmaI & XmnI double digestion, gel purification of the large (~8.9kb) backbone fragment, and self-ligation. Such a vector might be called pCAMBIA0381Z, but we have never constructed it for distribution as part of the pCAMBIA vector kit. If you have created a pCAMBIA vector derivative that other researchers will find useful, please let us know.