gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Plant Gene Expression Agrobacterium Binary Vector
Agrobacterium-mediated genetic transformation using binary vectors is a powerful and effective method for generating transgenic plants. This system utilized the ability of the bacteria Agrobacterium tumefaciens to insert foreign DNA into the genome of cells of numerous plant species.
The Agrobacterium binary vector system is derived from natural tumor-inducing (Ti) plasmids. Agrobacterium transfers a region of the Ti-plasmid known as the transfer DNA (T-DNA) into numerous plant species, where it is integrated into the host genome. The T-DNA is delineated by flanking 25 bp T-DNA border repeat sequences, in direct orientation with one another. In our binary vector system, all tumor-associated intervening T-DNA sequences have been removed, leaving the T-DNA border repeats, which flank and direct host integration of the user’s sequence of interest.
The complete binary vector system consists of two parts. The first, referred to as the T-DNA binary vector (or simply ‘binary vector’), contains two T-DNA repeats bracketing the DNA sequence which will be inserted into the plant host. The user’s gene of interest is cloned into this portion of the binary vector. The second plasmid, referred to as the vir helper plasmid, encodes components necessary for integration of the region flanked by the T-DNA repeats into the genome of plant cells. Prior to transforming the target plant cells, these two plasmids are brought together in Agrobacterium tumefaciens by co-transformation, co-electroporation, or conjugation.
When the binary vector and the vir helper plasmid are both present in the same Agrobacterium cell, proteins encoded by the vir helper plasmid act in trans on the T-DNA border repeat elements to mediate processing, secretion, and host genome integration of the sequence between the left and right border repeat elements. Insertion occurs without any significant bias with respect to insertion site sequence.
For further information about this vector system, please refer to the papers below.
|Plant Physiology. 146:325-32 (2008)||Review of T-DNA binary vector systems.|
|Trends in Plant Sci. 5:446-51 (2000)||Review of T-DNA binary vector systems.|
Our Agrobacterium binary vector system enables efficient insertion of sequences into genomes of target plant cells. The binary vector plasmid is optimized for replication in E. coli and Agrobacterium, efficient integration of user selected sequences into target plant cells, and high-level expression of transgenes.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of episomal DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transformation of plant cells with Agrobacterium vectors can deliver genes permanently into host plant cells due to the integration of the T-DNA region into the host genome.
Technical simplicity: Transformation of Agrobacterium with binary vectors is technically straightforward, as is transformation of plant cells using binary vectors and Agrobacterium.
Very large cargo space: Our Agrobacterium binary vector system can accommodate very large DNA inserts. Generally, inserts up to 20kb can be efficiently cloned transformed into target cells.
3’ deletions: Within the plant, it is common for nucleolytic degradation to delete sequence from the T-DNA left boundary (e.g. 3’) end. However, this is generally not a significant concern since the user’s sequence of interest is cloned near the right boundary, so degradation from the left boundary loss affects the marker gene.
Matching Agrobacterium strains and markers: Care must be taken to select Agrobacterium strains that work effectively with the specific binary vector and markers being used. For example, some Agrobacterium strains already express resistance to certain antibiotics, and binary vectors with these selection markers cannot be used in these strains.
Integration of backbone sequences: In some cases, integration of vector backbone sequences may occur along with T-DNA boundary-flanked sequence. This phenomenon occurs less frequently when low copy Agrobacterium plasmids are used, such as in our binary vector system.
Promoter: The promoter driving your gene of interest is placed here.
Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
Nos pA: The nopaline synthase polyadenylation signal of Agrobacterium tumefaciens. This facilitates transcription termination of the upstream ORF.
RB T-DNA repeat: Right border repeat of T-DNA. Upon recognized by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.
pVS1 StaA: Stability protein from the plasmid pVS1. Essential for stable plasmid segregation in Agrobacterium.
pVS1 RepA: Replication protein from the plasmid pVS1. Permits replication of low-copy plasmids in Agrobacterium.
pVS1 oriV: Origin of replication from the plasmid pVS1. Permits replication of low-copy plasmids in Agrobacterium.
pBR322 ori: pBR322 origin of replication. Facilitates plasmid replication in E. coli. Plasmids carrying this origin exist in low copy numbers (15-20 per cell) in E. coli if Rop protein is present, or medium copy numbers (100-300 per cell) if Rop protein is absent.
Kanamycin: Kanamycin resistance gene. It allows the plasmid to be maintained by kanamycin selection in bacterial hosts.
LB T-DNA repeat: Left border repeat of T-DNA. Upon recognized by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.
CaMV 35S_enhanced: A strong chimeric promoter which drives marker expression.
CaMV 35S pA: Cauliflower mosaic virus 35S polyadenylation signal. This facilitates transcription termination and polyadenylation of the marker gene.
Marker: A drug selection gene, allowing selection of plant cells transduced with the vector.