Cloning Vectors Characters and Types
Characteristic features of Vectors
Vectors used to produce the copies of desired genes by inserting into the host cell are called host vectors. These are also called DNA vehicles.
- Vector should have one or more unique restriction sites for restriction enzyme recognition.
- When foreign DNA fragment is inserted,there should be no change in its replication.
- It is easy to isolate from the host cell.
- They should have selectable maker genes resistant to antibiotics (ampicillin and tetracycline).
- Most importantly vectors should have ori genes for replication.
- Vectors should have tetra - genes which can transport in to host cells.
Types of Vectors
Plasmids are extra chromosomal, self replicating, circular naked DNA molecules found in the cytoplasm of bacteria. They are about one kbp to 500kbp in length. Plasmids are two types. They are
I) Conjugated plasmids
II) Non conjugated plasmids
As conjugated plasmids have transfer genes they transfer from one bacterium cell to another bacterium cell during conjugation. Non conjugated plasmids do not have transfer genes. There fore they cannot transfer from one cell to the other. In some bacteria the plasmid number is stable (1 or 2).These plasmids are called stringent plasmids. In some bacteria the plasmid number is more (10-100).These are known as relaxed plasmids.
In plasmids pBR322 is best example. This plasmid was first constructed by Bolivar (B) and Rodriguez (R). The plasmid has a point of origin of replication (ori), two selectable markers genes conferring resistance to antibiotics, eg., ampicillin (amp), tetracycline (tet) and unique recognition sites for 20 restrictions endo nucleases.
Bacteriophages are the Viruses that infect bacteria. These phages are being employed in gene cloning. 50%of the phage DNA is essential for its. Replication and other functions. Remaining 50%DNA can be replaced by desired genes.
M13 bacteriophage has single stranded DNA which infects E.coli cells. M13 Bacteriophage synthesis complementary DNA strand in host cell. This is called as replicated form of DNA. This DNA is isolated and covered into recombinant DNA by inserting the foreign DNA fragment it is introduced in to host cell as hybrid plasmid.
Smith and Townsend (1907)postulated that agrobacterium tumescent infects wounded or damaged plant tissues, induces the formation of of a plant tumer called crown gall. The entry of the bacterium into the plant tissues is facilitated by the release of certain phenolic compound aceto by the wound sites. Crown gall formation occurs when the bacterium releases its Ti plasmid into the plant cell cytoplasm. A fragment of Ti plasmid, reffer to as t-DNA, is actually transferred from the bacterium in to the host where it gets integrated into the plant cell chromosome. Agrobacterium tumescent induces crown gall disease in dicot plants. These tumer cells have Ti plasmid.
The Ti plasmids exist as independent replicating circular DNA molecules with the agrobacterium cells. The length of this plasmid measured about 200 kb.
Ti-plasmid has four main regions
It measures about 35kbp in length. This region for biosynthesis of auxin, cytokinin and opine. These three genes are referred as onco genes as they are the determinants of the tumor phenotype. A set of 24kb sequences present on either side of T-DNA are also ferried to the plant cells.
Virulence region codes for proteins involved in T-DNA transfer. At least nine virulence gene operons have been identified vir A1, vir C1, vir D1, and vir E1.
Opine catabolism region :
This region codes for proteins involved in the uptake and metabolism of opines.
It is responsible for the origin of DNA replication.