Vectors and its types
Vectors and its types
- A vector is a circular DNA molecule capable of independent existence and replication as example plasmid and Virus.
- In case of plants agrobacterium plasmid are the most commonly used vectors.
- A tumefacieus has the ti plasmid while a reasons has the ri plus made this plasmid have similar general feature and can I interchange between the two species.
- Both the plasmid have risen which content jeans for or metabolism and phytohormone independence this region is transferred into The plant cell and is integrated into their genome.
- They have another reason called vir region which produce among other things a endonuclease essential for the exhibition transfer of the reason into plant cell.
- The genes to the transferred are placed within t-dna of this plasmid.
- Therefore this transformation of vector into plants two vectors are prepared by genetic modification.
- They are co integrated vector and binary vector.
Co integrated vector
- In the CO integrated vector gene transferred is integrated with in the t DNA of a resident ti plasmid forming and intermediate vector.
- These are also known as hybrid ti-plasmid.
- These factors were among the first types of modified and engineered.
- TI plasmid devised for agrobacterium mediated transformation but they are not widely used now a days.
- This vectors are constructed by homologous recombination of a bacterial plasmid with the t DNA reason of an endogenous ti plasmid in agrobacterium.
- Integration of the two plasmid requires a reason of homologous present in both.
- These vectors are necessary in this system:
Disarmed agrobacterium ti plasmid:
- In this ti plasmid the oncogenes located in the t DNA reason have been replaced by exogenous DNA.
- These vectors include:-
- The right border of the t DNA together with the phytohormone genes coding for cytokinin and oxygen are removed and replaced by a bacterial kanamycin resistance gene while the left border and a small part of the segment of the original t DNA referred to as left inside homology are left intact.
- The phytohormone genes are excised and substituted by part of pbr322 vector sequence.
- The left and right border sequence as well as the nopeline synthase gene of the ti plasmid covered.
- These are small pbr322 based plasmid containing the t DNA reason.
- They are used to overcome the problems derived from the large size of disarmed ti plasmid and their lake of unique restriction sites.
- Intermediate vectors are replicated in e coli and transferred into agrobacterium by conjugation.
- They can not replicate in a tumefaciens and therefore carry DNA segments homologous to the disarmed T DNA to permit recombination to form a CO integrated t DNA structure.
- These are small plasmids maintained in e coli that contain transfer and mobilization gene which allowed the transfer of the consideration deficient intermediate vectors into agrobacterium.
- Resulting co integrated plasmid made assembled by vitro manipulation normally contents:
- The vir genes
- The left and right t-DNA borders
- An exogenous DNA sequence between the two T DNA borders.
- Plant and bacterial selectable markers.
- Although co integrated vectors have been designed to allow site specific recombination based on the recombination system of the phase P1.
- Large size of CP Plus made it is very difficult to handle and also presence of oncogenes or tumors carrying genes.
- Therefore co integrate vector is engineered genetically to insert into the plant.
In this strategy both the t DNA with our gene of interest and we are reason are present in the same tractor used for transformation.
Making of co integrated vectors
- At first and intermediate vector is mad using e coli plasmid + reason + t DNA borders + origin of replication + pbr322 sequences.
- Second vector is a disarmed PT vector =gene of interest + some markers + pbr322 sequences.
- Both intermediate vector and disarmed has some sequence in common which is PBR 322 sequences.
- Therefore by homologous recombination co integration of two plasmid will take place within agrobacterium.
- Now we have a CO integrated vector that has the t DNA with our gene of interest within the t DNA borders and VIR region.
Binary vector: Transformation of tobacco plant using co integrated vector:-
- The binary vector system consists of 2 plasmid that are involved in gene transformation and both are able to replicate separately inside the agrobacterium cell.
- The first plasmid is a modified ti plasmid which provides the VIR functions in trance and second contain gene of interest between the t DNA borders.
- Disarmed helper ti plasmid have been engineered by removing the oncogenic jeans while still providing the necessary VIR gene product required for transferring the t DNA to the host plant cell.
- A pair of plasmid consists of binary plasmid and helper plasma and are used to produce genetically modified plants.
- They are artificial vectors that have been created from the naturally occurring ti plasmid found in agrobacterium tumefaciens.
- The binary vector is a shuttle vector so called because it is able to replicate in multiple hosts.
- System in which t DNA and genes are located on a separate replicons are called t-DNA binary system.
- t DNA is located on the binary vectors.
- the replicon containing the VIR genes become known as VIR helper.
- There are several binary vector systems that differ mainly in the plasmid region that facilitates replication in agrobacterium.
- Commonly used binary vectors include PBINI9, PBVP, P GREEN.
Development of binary vectors:
- The PBINI9 plasmid was developed in the 1980 and is one of the first and most widely used to binary vector plasmid.
- P GREEN plasmid which was developed in 2000 is a newer version of the binary vectors that allow for choice of promoters selectable markers and reporter genes.
- Another distinguish feature of P GREEN plasmid is it’s large reduction in size PBINI9 there for increasing its transformation efficiency.
- Along with higher transformation efficiency P green has been engineered to ensure your transformation integrity.
- Due to polarity difference in the left and right borders the right border of the t DNA enters the host plant first.
- If the selectable marker is near the right border and the transformation process is interrupted the resulting plant may have expression of a selectable marker but contain no t-DNA giving of false positive.
- The p green plasmid has the selectable marker entering the host last so that any expression of the marker will result in full transgene integration.