Segregating population of progeny from Nicotiana
transformed with
Sinorhizobium meliloti containing
pCAMBIA1105.1R.
Photo taken by Dr Brian Weir, CAMBIA.
Below is a list of materials which are available under the Transbacter Project. They include live bacterial strains transformed with either unitary and binary vectors, as well as CAMBIA's unitary vectors. (The binary vectors are available under the GUSPlus Project.)
Rhizobium leguminosarum bv. Trifolii strain ANU845 + pCAMBIA5105 (Km and Spec selection)
Rhizobium leguminosarum bv. Trifolii strain ANU845 + pCAMBIA5106 (Km and Spec selection)Sinorhizobium meliloti + pCAMBIA 5105 (Km and Spec selection)
Sinorhizobium meliloti + pCAMBIA 5106 (Km and Spec selection)
Mesorhizobium loti + pWBTi1 + pCAMBIA1105.1R (Km and Spec selection)
Rhizobium sp. NGR 234 + pWBTi1 + pCAMBIA1105.1R (Km and Spec selection)
Rhizobium sp. NGR 234 + pWBTi3 + pCAMBIA1105.1R (Km and Spec selection)
Sinorhizobium meliloti + pWBTi1 + pCAMBIA1105.1R (Km and Spec selection)
Sinorhizobium meliloti + pWBTi3 (Km selection)
Sinorhizobium meliloti + pWBTi3 + pCAMBIA1105.1R (Km and Spec selection)Culturing information for Transbacter strains (info)
View General and Import Information (info)
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.
Click here to view pCAMBIA5105 plasmid map
pCAMBIA5106
pCAMBIA5106 is yet a reduced version of pCAMBIA5105. It lacks virA and virK and has a virG(N54D) mutation in the virG gene. It is smaller than pCAMBIA5105, and because it is virA independent, there is no need to use acetosyringone and seems to be working very well in rice.
View pCAMBIA5106 plasmid map.
TransBacter™ focuses on the use of bacteria outside the genus Agrobacterium for gene transfer to plants.
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Segregating population of progeny from Nicotiana
transformed with |
Since the discovery in the 1970's that Agrobacterium tumefaciens is capable of transferring genes to plants, it has become the most important tool in plant biotechnology. It is also widely considered to be the only bacterial genus capable of transferring genes to plants. We have shown that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to diverse plants. Bacteria from two families, and three genera, Rhizobium sp. NGR234, Sinorhizobium meliloti and Mesorhizobium loti, were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a binary vector (info). Stable transformation of three plant species, tobacco, rice and Arabidopsis was achieved with these non-Agrobacterium species.
Transformation was performed using minor modifications of published protocols, using leaf disk (info), scutellum-derived callus (info) or floral dip (info) methods, respectively. The resulting plants expressed hygromycin resistance and GUS activity, contained 1-3 copies of the T-DNA by Southern blot analysis, and showed the normal range of T-DNA insertions into host genomes by PCR-mediated sequencing of integration sites. To ensure that the gene transfer did not result from contamination with Agrobacterium cells, controls including species-specific PCR (info), selective plating (info), and use of a tagged binary vector (info) were implemented.
Thus, diverse plant-associated bacteria, when harbouring a disarmed Ti plasmid and binary vector (or presumably a co-integrate or whole Ti plasmid), are readily able to transfer T-DNA to plants. The Ti plasmid is self-transmissable, perhaps indicating the existence of a ubiquitous natural mechanism effecting horizontal gene transfer from bacteria to plants. If so, then much of the plant genomic DNA sequence thus far determined, which seems bacterial in origin, may have derived from such transfer over recent evolutionary timescales.
This alternative to Agrobacterium-mediated technology may provide two distinct advantages:
This new gene transfer technology is freely available here through this novel open-source inspired BIOS license that will encourage ethical, shared and transparent development and use of the technology (Nature 431: 494, 2004). It is envisaged that this will help to break the dominance of plant transformation by a few multinational companies and will forge a highly cooperative and public-spirited technology development process. This step towards independence from corporate control of enabling technology may foster diverse applications of biotechnology to emerge with a focus on public good and low-margin priorities typical of the needs of developing world agriculture, production environments and economies.
Broothaerts W, Mitchell H J, Weir B, Kaines S, Smith L M A, Yang W, Mayer J E, Roa-Rodriguez C, Jefferson R A (2005) Gene transfer to plants by diverse species of bacteria, Nature 433:629-633.
General
- Gelvin S B (1998) The introduction and expression of transgenes in plants, Curr. Op. in Biotechnol., 9:227-232.
- Gelvin S B (2000) Agrobacterium And Plant Genes Involved In T-DNA Transfer And Integration, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:223-56.
- Gelvin S B (2003) Improving plant genetic engineering by manipulating the host, TRENDS in Biotechnology, 21:95-98.
- Gelvin S B. 2003 Agrobacterium-Mediated Plant Transformation: the Biology behind the 'Gene-Jockeying' Tool, Microbiology And Molecular Biology Reviews, 67: 16-37.
- Zhu J, Oger P M, Schrammeijer B, Hooykaas P J J, Farrand S K, Winans S C (2000) The Bases of Crown Gall Tumorigenesis, J. of Bacteriol., 182: 3885-3895.
Conjugation and secretion systems
- Christie P J (2004). Type IV secretion: the Agrobacterium virB/D4 and related conjugation systems. Biochem. Biophys. Acta. 1694:219-234.
- Gauthier A, Thomas N A, Finlay B (2003) Minireview: Bacterial Injection Machines, J. Biol. Chem., 278:25273-25276.
- Hubber A, Vergunst A C, Sullivan J T, Hooykaas P J J, Ronson C W (2004) Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system Molecular Microbiology 54(2):561-574.
- Lees M, Lanka E (1994) Common Mechanisms in Bacterial Conjugation and Ti-Mediated T-DNA Transfer to Plant Cells, Cell, 77:321-324.
- Llosa M, de la Cruz F (2005) Bacterial conjugation: a potential tool for genomic engineering, Research in Microbiology 156:1-6
- O'Callaghan D, Cazevielle C, Allardet-Servent A, Boschiroli M L, Bourg G, Foulongne V, Frutos P, Kulakov Y, Ramuz M (1999) A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis PtI type IV secretion systems is essential for intracellular survival of Brucella suis, Molecular Microbiology, 33(6):1210-1220.
- Schröder G, Lanka E (2005) Review: The mating pair formation system of conjugative plasmids - A versatile secretion machinery for transfer of proteins and DNA, Plasmid, 54:1-25.
- Teyssier-Cuvelle S, Mougel C, Nesme X (1999) Direct conjugal transfers of Ti plasmid to soil microflora Molecular Ecology 8:1273-1284.
- Teyssier-Cuvelle S, Oger P, Mougel C, Groud K, Farrand S K, Nesme X (2004) A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems Microbial Ecology 48:10-18.
- Viprey V, Del Greco A, Golinowski W, Broughton W J, Perret X (1998) Symbiotic implications of type III protein secretion machinery in Rhizobium, Molecular Microbiology 28(6):1381-1389.
- Waters, V.L. 1999 Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance, Frontiers in Bioscience, 4:d433-456.
- Weller S A, Stead D E, Young J P W(2004) Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other a-proteobacteria in cucumber and tomato crops affected by root mat Applied and Environmental Microbiology 70(5):2779-2785.
vir Regulon
- Hamilton C M, Hyewon L, Pei-Li Li, Cook D M, Piper K R, Beck Von Bodman S, Lanka E, Ream W, Farrand S K (2000) TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58, Journal of Bacteriology 182:1541-1548.
- Kalogeraki V S, Winans S C (1998) Wound-Released Chemical Signals May Elicit Multiple Responses from an Agrobacterium tumefaciens Strain Containing an Octopine-Type Ti Plasmid, Journal Of Bacteriology, 180:5660-5667.
- Krishnamohan A, Balaji V, Veluthambi K (2001) Efficient vir Gene Induction in Agrobacterium tumefaciens Requires virA, virG, and vir Box from the Same Ti Plasmid, Journal of Bacteriology, 183:4079-4089.
- Lessl M, Lanka M (1994) Common Mechanisms in Bacterial Conjugation and Ti-mediated T-DNA transfer to plant cells, Cell, 77:321-324.
- Pazour G J, Anath D (1990) virG, an Agrobacterium tumefaciens Transcriptional Activator, Initiates Translation at a UUG Codon and Is a Sequence-Specific DNA-Binding Protein, Journal of Bacteriologyy,172:1241-1249.
- Schrammeijer B, Beijersbergen A, Idler K B, Melchers L S, Thompson D V, & Hooykaas P J J (2000) Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955, J. Exp. Bot., 51:1167-1169.
- Schröder G, Lanka E (2005) Review: The mating pair formation system of conjugative plasmids A versatile secretion machinery for transfer of proteins and DNA, Plasmid, 54:1-25.
Genetic Tools
- Ditta G, Stanfield S, Corbin D, Helinski D R (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti, Proc. Natl. Acad. Sci., 77:7347-7351. (RK2 and pRK290)
- Scott H N, Laible P D, Hanson D K (2003) Sequences of versatile broad-host-range vectors of the RK2 family, Plasmid, 50:74-79.
- Lohrke S M, Yang H, Jin S (2001) Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli, Journal of Bacteriology, 183:3704-3711.
See Also Frequently Asked Questions about Request Procedures or download the TransBacter Request Form (info).
TransBacter is a collective name given to non-pathogenic bacteria that have been modified to be able to replace Agrobacterium in plant transformation processes. To date we have shown, using modified plasmids, that three species of bacteria, Sinorhizobium meliloti, Mesorhizobium loti, and Rhizobium sp. NGR234 are able to transfer genes to plants under certain circumstances.
TransBacter strains have been made available to the scientific community through a BiOS License. To use TransBacter strains you must agree to the terms of the BiOS Plant Enabling Technology License (info).
By using TransBacter it may be possible to circumvent the current licensing thicket involved with using Agrobacterium for plant transformation (see Agrobacterium-mediated Transformation of Plants Technology Landscape for more information).
We provide TransBacter strains for research by non-profit organisations, or for-profit companies. To obtain TransBacter strains for research in non-profit organisations, simply complete the TransBacter Request Form (info)and send it to us by mail or fax. We'll send the strains as soon as our lab commitments permit it, and if you are located in an OECD country a small charge will be made to cover replication and mailing costs.
To request a license for use within a company or commercial environment, contact us at licenses@cambia.org. The company will be asked to execute a BiOS Technology Support Agreement to help us cover our costs in protecting the technology and supporting the website and materials to provide the technology and improvements.
You may need to find out about requirements for importing these bacteria into your country. CAMBIA doesn't accept any liability for checking these because each jurisdiction and institution may have different rules, and complying with these is the responsibility of the recipient. However, if you need information to fill out forms, you may be able to find it on this website (see below) or ask colleagues from your country through our discussion forum. We encourage people who have imported these bacteria to post the information they used.
The patent applications and trademark 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 TransBacter. 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". An entity may obtain a license to use the technology only by agreeing to all the terms of the license, including allowing other licensees to use improvements and biosafety data. An entity that attempts to enforce patents on the improvements against others voids its license and would be liable for infringement.
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.
To provide non-profits with TransBacter strains and the licenses to use them, the only payment required is to recover our direct costs in replicating and sending out the strains. Please see the TransBacter request form for current charge information. In special circumstances, especially for labs not located in OECD countries, we may waive even this charge.
For-profit companies are requested to enter into a subscription agreement that supports the development of the protected commons, with annual fees commensurate to the size of the company and where it is based.
All BiOS licenses are completely royalty-free.
We provide licenses to use TransBacter strains for both for-profit business or non-profit research.
Copies of both the BiOS License for Plant Enabling Technologies (info) and the attached Technology Support Agreement are available at BiOS website.
In signing a TransBacter Request Form and/or using TransBacter strains you agree to the conditions of these agreements.
BiOS licenses are available from CAMBIA completely royalty-free for both 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 support 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 and non-profits obtain all the benefits of the license without any such subscription.
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.
Very few companies make a profit on patent licenses! Patents are expensive to file and maintain. CAMBIA doesn't try to make a profit with its licenses. We are a non-profit organisation. All funds go to support CAMBIA's initiatives to make capabilities to use technology widely accessible.
You can register your details on the BioForge.
Registering with BioForge gives you the protected availability and opportunities to discuss technical issues and improvements and to share results, ideas, observations, questions and data with fellow BiOS licensees.
Royalty-free licenses are available from CAMBIA to both for-profit and non-profit entities. CAMBIA believes that hunger in many parts of the world can be alleviated not only by providing tools to public good researchers in those communities, but also by removing barriers to micro, small and medium enterprise.
IBM, Nokia, Sun and other IT companies have shown that hardware and software technology can be non-exclusively and open-source licensed and simultaneously generate profitability in a for-profit company. BiOS-compliant licenses make it possible to do the same for life sciences products.
If you are working in a non-profit or university, fill out and sign the TransBacter Request Form (info) and send it to us. Please note on the form who will supply you with the strains, and cross out the payment section (as the charges are solely to cover production and distribution, payment to cover postage is not required if obtaining strains from a friend or colleague). Once you have registered your agreement to the conditions of the license, you will have a license to use strains from your colleague.
If you are working in a for-profit company or on a commercial project, the company should execute a technology support services subscription agreement to help us cover our costs in making the technology and improvements available, both through materials support and this website.
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The terms of the BiOS license require users of Transbacter to comply with applicable laws. Every jurisdiction has different rules. You will need to find out if there are any importation/custom requirements for mailing of these strains to your country.
Once you have obtained the appropriate forms, these frequently asked questions and other information on the website should give you enough information to fill out the forms. We encourage people who have imported these bacteria to post the information they used.
For example, see General Information about TransBacter (info) which contains the information that was required by a recipient within the US to successfully obtain a USDA-APHIS permit.
When you provide us with the appropriate documentation, we will attach it to the package to send to you. If you don't need such documentation for your country, please advise us of that on your request form.
Depending on your request, the kit will contain bacterial strains (e.g. Sinorhizobium meliloti, Rhizobium sp. or Mesorhizobium loti). Each bacterial strain is individually streaked onto 1.5mL of YM Agar medium in a sealed plastic tube.
The number of tubes containing bacteria on media depends on the number of bacterial strains requested.
You will also receive a BioForge registration card, CD containing TransBacter information such as vector maps, and instructions on culturing the TransBacter strains.
pCAMBIA1105.1R is specifically provided to use with TransBacter strains. The "R" signifies the presence of a multiple cloning site with a size marker that distinguishes it from pCAMBIA1105.1. Some variants of this vector are now available. pCAMBIA1105.1 is intended to use with Agrobacterium controls. A description and maps of these vectors can be downloaded here.
The strains we are distributing are modified in that they contain a derivative of the pTiBo542 plasmid from Agrobacterium tumefaciens. pTiBo542 had been "disarmed" by having both of its T-DNA regions deleted, to create pTiEHA101 (with an nptI gene in place of the deleted T-DNA) and pTiEHA105 (with no foreign DNA inserted in place of the deleted T-DNA). For details, see Hood et al 1986 J Bact 168:1291-1301 and Hood et al 1993 Trans Res 2:208-218.
pTiEHA105 was modified at CAMBIA by homologous recombination insertion of a pCR2.1TOPO plasmid (Invitrogen Inc.) containing an RK2 oriT (amplified from pSUP202 with primers EVS49 and EVS50 - see Stabb and Ruby 2002 Methods Enzymol. 358:413-426) and the virG gene - to create pTiWB1 - or the moaA gene - to create pTiWB3.
Some strains also contain the binary vector pCAMBIA1105.1R with a GUSPlus reporter construct (see the GUSPlus Project).
At this time only Sinorhizobium meliloti + pWBTi3 can be requested without the binary vector pCAMBIA1105.1R. Other strains and combinations may be available later.
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In both cases the strain was first transformed with pCAMBIA1105.1R by electroporation and the modified Ti plasmids pWBTi1 or pWBTi3 were introduced next by triparental mating from Sinorhizobium meliloti.
You should be able to cure these bacteria of the binary plasmid by growth in non-selective conditions (no spectinomycin or streptomycin) for some generations, followed by replica plating on media with and without spec (or strep) to screen for colonies that have become sensitive to it.
A quicker method would be to transform in another pCAMBIA plasmid (or other plasmid with the same backbone such as the pPZP plasmids) and select for its resistance gene. This should "bounce out" pCAMBIA1105.1R since cells don't like to keep two different plasmids with the same replicon. You should still screen for loss of spec/strep resistance though, to make sure.
We don't intend to do this ourselves. We are busy working on a broad host range Ti plasmid and this will be made available in strains without the presence of a binary plasmid.
We have experience using TransBacter to transform tobacco, Arabidopsis and rice. Other labs have had similar success and are trying other species. We encourage you to use TransBacter on the organism that you are researching.
Your state or country may require permits for the strains. The conditions of the BiOS license require that recipients comply with all applicable laws.
We try whenever possible to refer requestors to a BiOS Reference Lab in the same nation for the redistribution of materials. Depending on the applicable laws in your nation, this may help to minimise import and quarantine restrictions. If you would like to be a BiOS Reference Lab in your country to replicate and send out the strains on our behalf, please e-mail us. We would provide you with the mailing kits, including CDs with media compositions, protocols, copies of the license, etc.
For more information about how you can help, use the links on the left under "How do I contribute?"
If you wish to obtain any of the Materials offered by CAMBIA please follow the instructions below:
Frequently asked questions:
In return for the benefits of the technologies, a licensee institution agrees to allow and encourage its employees and students to post any improvements made to the technologies and safety information relevant to use of the technology and potential regulatory approval of products embodying it, and agrees not to assert any intellectual property rights to the improvements and information against other licensees.
The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.