pCambia Vectors

Information on pCAMBIA5105 may be found below, as well as in the BioForge Transbacter Project.

Information on our latest vectors, which contain GUSPlus™, may be found in the BioForge. You can request these vectors separately (they are available under the open source terms of a BiOS license (info) ) or simultaneously when registering for the TransBacter Project , in which we're improving the alternative to Agrobacterium transformation for more complete freedom to operate and freedom to cooperate.

Much of the information below relates to older pCAMBIA vectors, which will soon be available under a new MTA with similar conditions to facilitate cooperative development.

Description of the pCAMBIA vectors
Quick-pick table and Genbank links
Description of pCAMBIA vectors

pCAMBIA5105
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. These methods have both technical and intellectual property limitations (see the BioForge TransBacter Project , in 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:

low-copy origin of replication resulting in low yield DNA preps;
unstable replicons, causing variable loss of the plasmid during propagation;
large size;
lack of convenient restriction sites for manipulation;
limited choice of selectable markers for both bacteria and plants;
lack of simple ways to construct reporter gene fusions;

The pCAMBIA vector backbone is derived from the pPZP vectors (constructed by Hajdukiewicz, Svab & Maliga, see References). While not perfect and having technical and IP limitations (see the BioForge GUSPlus Project , in which we're improving reporter genes and selection methods for more complete freedom to operate), pCAMBIA vectors offer:

high copy number in E.coli for high DNA yields
pVS1 replicon for high stability in Agrobacterium
small size, 7-12kb depending on which plasmid
restriction sites designed for modular plasmid modifications and small but adequate poly-linkers for introducing your DNA of interest
bacterial selection with chloramphenicol or kanamycin
plant selection with hygromycin B or kanamycin (phosphinothricin selection was discontinued at the request of the IP owner, Bayer, after the initial distribution in 1997)
simple means to construct translational fusions to gusA reporter genes.

A few points about the pCAMBIA cloning strategy:

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 SphI (which has an ATG) or XbaI (which has a TAG). This makes other sites in the vector more useful (such as the SphI site outside the right T-DNA Border, or the SacII 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 NheI site (there is a second NheI site in the pVS1-rep that we didn't eliminate) and the unique PmlI site. Genes of interest may be inserted in place of the reporter gene. Insertion without a stop codon and in frame at the (first) NheI site will append a hexa-Histidine tag to your protein of interest. Insertion without a stop codon and in frame at the PmlI site will append a stop codon. Insertion at the BstEII 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).

GeneralStructure

Nomenclature of pCAMBIA vectors:

The four digit numbering system works as follows:

First digit - indicates plant selection: 0 for absence; 1 for hygromycin resistance; 2 for kanamycin; and 3 for phosphinothricin (the vectors containing the phosphinothricin resistance gene are no longer available from CAMBIA at the request of Bayer, which owns patents restricting its use in some countries).

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 - notes some other special feature. So far this has been used only with: pCAMBIA1305.1 and plasmids derived from it, where the .1 denotes the absence of a signal peptide from the GUSPlus™ protein; and pCAMBIA1305.2 where the .2 denotes the presence of the GRP signal peptide for in planta secretion of the GUSPlus™ protein.

Lagging letter - X indicates that the reporter gene lacks its own start codon and the vector is for creating fusions to the reporter; Z indicates presence of a functional lacZa for blue-white screening; a/b/c indicates the reading frame for fusions with the Fuse and Use vectors.

Important note: 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 pCAMBIA2205.2. 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.

Vector Manual Addendum: Important note to pCAMBIA users

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

pCAMBIA	R-gene plants	R-gene bacteria	Polylinker	Reporter Gene	T-DNA size (bp)	Reading Frame or lacZ	Vector family	Map
5105	hpt`II`	kan+spec/strep		gus plus	5599	lacZ	GIS	view map
2301	npt`II`	kan	pUC18	gusA	5391	-	GIS	view map
2300	npt`II`	kan	pUC18	-	2512	-	MSV	view map
2201	npt`II`	cmr	pUC18	gusA	5391	-	GIS	view map
2200	npt`II`	cmr	pUC18	-	2512	-	MSV	view map
1405.1	hpt`II`	kan+ spec/strep	pUC18	gus plus	5593	lacZ	GIS	view map
1391	hpt`II`	kan	pUC9	gusA	4427	-	FUV	view map
1391Z	hpt`II`	kan	pUC9	gusA	4984	lacZ	PCV	view map
1391Xc	hpt`II`	kan	pUC9	gusA	-	c	FUV	view map
1391Xb	hpt`II`	kan	pUC9	gusA	-	b	FUV	view map
1391Xa	hpt`II`	kan	pUC9	gusA	-	a	FUV	view map
1390	hpt`II`	kan	pUC9	-	2630	-	MSV	view map
1381	hpt`II`	kan	pUC8	gusA	4427	-	FUV	view map
1381Z	hpt`II`	kan	pUC8	gusA	4984	lacZ	PCV	view map
1381Xc	hpt`II`	kan	pUC8	gusA	-	c	FUV	view map
1381Xb	hpt`II`	kan	pUC8	gusA	-	b	FUV
1381Xa	hpt`II`	kan	pUC8	gusA	-	a	FUV	view map
1380	hpt`II`	kan	pUC8	-	2630	-	MSV	view map
1305.2	hpt`II`	kan	pUC18	gus plus with secretion signal peptide	5667	-	GIS	view map
0305.2 (identical to above vector but contains no hpt`II` cassette)	-	kan	pUC18	gus plus with secretion signal peptide	3412	lacZ	GIS
1305.1	hpt`II`	kan	pUC18	gus plus	5592	-	GIS	view map
0305.1 (identical to above vector but contains no hpt`II` cassette)	-	kan	pUC18	gus plus	3408	lacZ	GIS	view map
1304	hpt`II`	kan	pUC18	gfp:gusA	6128	-	GFP	view map
1303	hpt`II`	kan	pUC18	gusA:gfp	6128	-	GFP	view map
1302	hpt`II`	kan	pUC18	gfp	4316	-	GFP	view map
1301	hpt`II`	kan	pUC18	gusA	5607	-	GIS	view map
1300	hpt`II`	kan	pUC18	-	2728	-	MSV	view map
1291Z	hpt`II`	cmr	pUC9	gusA	4984	lacZ	PCV	view map
1281Z	hpt`II`	cmr	pUC8	gusA	4984	lacZ	PCV	view map
1201	hpt`II`	cmr	pUC18	gusA	5606	-	GIS	view map
1200	hpt`II`	cmr	pUC18	-	2727	-	MSV	view map
1105.1 (download as attachment)	hpt`II`	spec/strep	pUC18	gus plus	5854	lacZ	GIS	view map
1105.1R (download as attachment)	hpt`II`	spec/strep		gus plus	5915	-	GIS	view map
0105.1R (download as attachment)	-	spec/strep		gus plus	3489	-	GIS	view map
0390	-	kan	pUC9	-	582	-	DIY	view map
0380	-	kan	pUC8	-	582	-	DIY	view map

pCAMBIA5105

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 1105.1 and a fragment of the Ti plasmid (derived from pTiBo542) containing only the virA, virB, virC, virD, virE, virG, virK & virJ operons. 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:
• It can be distributed as spots of dilute DNA on paper (e.g. on a letter) eliminating the need for compliance with importation controls on living organisms, and other quarantine issues. This is the means by which literally thousands of laboratories worldwide have obtained (and further sent out) pCAMBIA vector sets.
• GT-BacK vector lacks the RK2 derived oriT that Transbacter carried, thus eliminating or reducing the ability of the plasmid to be conjugated and transmissible to other hosts by RK conjugation functions.
• It has a broad host range replication origin from pVS1 allowing much wider spectrum of bacteria to be explored as gene transfer vectors, allowing choices of benign symbionts which do not impose physical or genetic stresses on plants.

If you wish to discuss or query any CAMBIA materials please login to the

Do-it-yourself vectors

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 BioForge 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 of Agrobacterium 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 BioForge 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 BioForge 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

pCAMBIA1201; pCAMBIA1301; pCAMBIA2201; pCAMBIA2301

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 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.

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.

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.

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 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 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.

Genotypes of some useful Agrobacterium tumefaciens strains

Warning!! 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.

LBA4404 (Ach5 pTiAch5) Sm/Sp(R) in the virulence plasmid (from Tn904); all T-DNA of pTiAch5 eliminated in pAL4404 (Hoekema et al., 1983).
EHA101, genotype C58 pTiBo542; T-region::aph, Km(R); A281 derivative harboring pEHA101, T-DNA replaced with nptII, elimination of T-DNA boundaries uncofirmed, super-virulent (Hood et al., 1986).
EHA105 is a Km(S) derivative of EHA101 (Hood et al., 1993).
AGL1, genotype is AGL0 (C58 pTiBo542) recA::bla, T-region deleted Mop(+) Cb(R) [AGL0 is an EHA101 with the T-region deleted, which also deletes the aph gene] (Lazo et al., 1991).
A281, reconstructed strain, derivative of A136 (cured C58) harboring pTiBo542, super-virulent (Hood et al., 1986).

Types

Ach5 .......... agrocinopine, octopine type
B6S3, A6 .... octopine type
Bo542 ........ leucinopine, succinamopine, agropine type, vir weaker than A281
C58, T37 .... nopaline types
A281 ......... succinamopine, leucinopine, agrocinopine

Antibiotics

Chloramphenicol, 100 µg/mL for strain AGL1, 10 µg/mL for LBA4404, 25 µg/mL for EHA105 and for E. coli.
Kanamycin, 50 µg/mL for both Agrobacterium and E. coli.
For selection of transformed rice plants we use hygromycin at 50 µg/mL and 25 µg/mL for tobacco.

Selected References

Chen L, Zhang S, Beachy RN, Fauquet CM (1998) A protocol for consistent, large-scale production of fertile transgenic rice plants. Plant Cell Reports 18:25-31

Christou P (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Biotechnology 9:957-962

Christou P (1997) Rice transformation: bombardment. Plant Mol Biol 35:197-203.

Deblaere R, Reynaerts A, Hofte H, Hernalsteens JP, Leemans J, and Van Montagu M (1987) Vectors for cloning in plant cells. Meth Enzymol 153:277-292

Hajdukiewicz,P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994

Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271-282

Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Binary vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179-180

Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bac 168:1291-1301

Hood EE, Gelvin SB, Melchers S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants (EHA105). Trans Res 2:208-218

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901-3907

Klapwijk PM, van Breukelen J, Korevaar K, Ooms G, Schilperoort RA (1980) T ransposition of Tn904 encoding streptomycin resistance into octopine Ti plasmid of Agrobacterium tumefaciens. J Bac 141:129-136

Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Ara bidopsis genomic library in Agrobacterium. BioTechnology 9:963-967

Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a beta-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805-814

Ooms G, Hooykaas PJJ, Van Veen RJM, Van Beelen P, Regensburg-Tunk JG, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7 :15-29

Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J 5:1137-1142

Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expre Purif 3:263-281

Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol 6:1653-1663

Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K (1990) 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 18:6767-6770

Other Resources

If you wish to discuss or query any CAMBIA materials please email us at materials@cambia.org
Read Frequently Asked Questions about requesting CAMBIA materials
Read Frequently Asked Questions about pCAMBIA Vectors
GUSPlus information in the BioForge GUSPlus Project .

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