Sign up for updatespBrachyTAG vector system
The aim of this project is to develop an improved binary vector system, pBrachyTAG, for T-DNA tagging and promoter trapping in Brachypodium distachyon. While genetic and genomic resources are rapidly being developed for Brachypodium (including the release of the complete genome sequence in 2009, large collections of T-DNA insertion lines remain to be produced. The pBrachyTAG vector system enables gene tagging and trapping in Brachypodium with an improved efficiency and is available as a community resource. This project is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) - Tools and Resources project CA351.
1. Description of pBrachyTAG vector system
The pBrachyTAG vectors are based on the pCLEAN (Thole et al., 2007 Plant Phys.145:1211-1219) dual binary vector system and are designed to address current limitations in characterising flanking plant genomic sequences and creating functional gene fusions in promoter trapping strategies. These new vectors contain features known.
- to reduce vector backbone transfer
- to limit the introduction of superfluous DNA sequences
- to improve transformation efficiency
- to enable more than one PCR-based FST retrieval strategy using universal sequences (UniBox system).
The complete sequence of the pBrachyTAG-G217 (GenBank acc. GU947644) and pBrachyTAG-G218 (GenBank acc. GU947643) vectors are available from GenBank. All pBrachyTAG vectors were assembled using the basic dual binary vector pCLEAN-G181 (GenBank acc. EU186083). Additional vectors containing a different terminator (-G220, -G221), reporter gene (-G229) and/or unique restrictions sites for promoter/gene-of-interest studies (-G225, -G226, -G227) have also been constructed and are being tested.
2. Using pBrachyTAG vectors
The pBrachyTAG vectors are compatible with the pCLEAN and pGreen/pSoup dual binary vector systems, where two binary vectors (i.e. pGreen+pSoup or pCLEAN-G+pCLEAN-S) are both present in the same Agrobacterium. In Agrobacterium, pSoup provides replication function in trans for the pGreen-based vectors. Both pGreen-based and pSoup-based vectors can be maintained individually in Escherichia coli.
3. Ordering pBrachyTAG vectors
The pBrachyTAG vector system (including pBrachyTAG-G217, -G218 and basic pClean-S constructs) is available as a community resource and can be obtained by scientists on a cost-recovery basis. The fee per order is £40 (payable by credit card). A Material Transfer Agreement form and Credit Card Order form are available from Vera Thole or Philippe Vain.
Dr. Vera Thole |
Dr. Philippe Vain |
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