Vectors
Another major component of a gene cloning experiment is a vector such as a plasmid. A Vector is a small DNA molecule capable of self-replication and is used as a carrier and transporter of DNA fragment which is inserted into it for cloning experiments. Vector is also called cloning vehicle or cloning DNA.
Vectors are of two types:
i) Cloning Vector, and
ii) Expression Vector.
Cloning vector is used for the cloning of DNA insert inside the suitable host cell. Expression vector is used to express the DNA insert for producing specific protein inside the host. Properties of Vectors Vectors are able to replicate autonomously to produce multiple copies of them along with their DNA insert in the host cell.
• It should be small in size and of low molecular weight, less than 10 Kb (kilo base pair) in size so that entry/transfer into host cell is easy.
• Vector must contain an origin of replication so that it can independetly replicate within the host.
• It should contain a suitable marker such as antibiotic resistance, to permit its detection in transformed host cell.
• Vector should have unique target sites for integration with DNA insert and should have the ability to integrate with DNA insert it carries into the genome of the host cell.
Most of the commonly used cloning vectors have more than one restriction site. T hese are Multiple Cloning Site (MCS) or polylinker. Presence of MCS facilitates the use of restriction enzyme of choice.
The following are the features that are required to facilitate cloning into a vector.
1 – Origin of replication (ori):
This is a sequence from where replication starts and piece of DNA when linked to this sequence can be made to replicate within the host cells.
2- Selectable marker:
In addition to ori the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.
3. Cloning sites:
In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
Types of vector
Few types of vectors are discussed in detail below:
Plasmid
Plasmids are extra chromosomal, self replicating ds circular DNA molecules, found in the bacterial cells in addition to the bacterial chromosome. Plasmids contain Genetic information for their own replication.
pBR 322 Plasmid
pBR 322 plasmid is a reconstructed plasmid and most widely used as cloning vector; it contains 4361 base pairs. In pBR, p denotes plasmid, Band R respectively the names of scientist Boliver and Rodriguez who developed this plasmid. The number 322 is the number of plasmid developed from their laboratory. It contains ampR and tetR two different antibiotic resistance genes and recognition sites for several restriction enzymes. (Hind III, EcoRI, BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes. Rop codes for the proteins involved in the replication of the plasmid.
Ti Plasmid Bacteria
Ti plasmid is found in Agrobacterium tumefaciens, a bacteria responsible for inducing tumours in several dicot plants. The plasmid carries transfer (tra) gene which help to transfer T- DNA from one bacterium to other bacterial or plant cell. It has Onc gene for oncogenecity, ori gene for origin for replication and inc gene for incompatibility. T-DNA of Ti-Plasmid is stably integrated with plant DNA. Agrobacterium plasmids have been used for introduction of genes of desirable traits into plants
