VECTORS
The term vector refers to the DNA molecules that act as transporting vehicle which carries foreign DNA into a host cell for the purpose of cloning and expression. Cloning vectors are used to clone foreign DNA whereas expression vectors are engineered so that any foreign DNA can be transcribed in RNA and translated into protein. A viral DNA or plasmid is generally used as a vector.
Important features of cloning vectors
- Ability to replicate in host cells.
- Unique restriction enzyme sites for insertional cloning.
- Genetic marker to select for host cells containing the vector.
- Low molecular weight.
Types of vector
Vectors are of two types:
1. Cloning vector
2. Expression vector
Cloning vectors
Cloning vectors are small piece of DNA which have the ability and used to introduce foreign gene of interest into the host cell.
They can be stably maintained insides the host cell.
Cloning vector are generally used to obtain multiple copies of desired foreign gene.
Example- Plasmid, Cosmid and Phages, BACs, YACs.
These type of vectors generally contains selectable marker, origin of replication and a restriction site.
Expression vector
Expression vector is a type of vector which not only introduces a gene of interest into the host cell but also aids in the analysis of the foreign gene via relevant protein product expression.
It is type of vector which is used to obtain or analyses the gene product, which may be RNA or protein of the inserted desired gene.
Example- Only plasmid vector.
Expression vector contains enhancer, promoter region, start/stop codon, transcription initiation, selectable marker, ori sites, and restriction site.
A. Plasmid vector
A plasmid is a small, circular piece of DNA that is different than the chromosomal DNA, which is all the genetic material found in an organism’s chromosomes. It replicates independently of chromosomal DNA. Plasmids are mainly found in bacteria, but they can also be found in archaea and multicellular organisms. Plasmids usually carry at least one gene, and many of the genes that plasmids carry are beneficial to their host organisms. Although they have separate genes from their hosts, they are not considered to be independent life.
Example: pBR322 , and pUC19.
pBR322
It is one of the first vectors to be developed by Boliver and Rodriguez in 1977.
The name pBR322 denotes the following:
P- plasmid
B- Boliver
R- Rodriguez
322- Differentiate it from the other plasmid produced in the same laboratory.
It is 4361 base pairs long.
It carries two sets of antibiotic resistance genes
• Ampicillin resistance gene.
• Tetracycline resistance gene.
pUC19
It is a derivative of pBR322.
It is 2686 base pairs long.
It caries two sets of genes
• Ampicillin gene
• lac Z gene
B. Cosmids
It is hybrid of lambda phages and plasmids.
Cosmids = cos sites + plasmid.
Genomic size of cosmids is about 30 to 52 kb.
If they have suitable origin of replication than they can replicate as Plasmid within the host cells.
C. Phagemid Vector
A plasmid vector which contains an origin of replication from a phage, in addition to that of the plasmid, is termed as Phagemids.
pBlue script SK (+/-) is a type of phagemid vector of 2,958 bp derived from the pUC19.
It consists of the following:
- ColE1 ori
- Phage f1 (M13) origin of replication.
- A small portion of lacZ gene.
- MCS within lacZ Gene from Lac promoter.
- Phage T7 and T3 promoter sequence.
- Ampicillin gene for Ampicillin resistance.
D. Phage vector or Bacteriophage vector
Bacteriophages are viruses that attacks bacteria.
The Phages are simple in structure.
It consists of DNA molecules having several gene for replication which is surrounded by Capsid.
On the basis of structure bacteria phases are of two types:
Head and Tail Phages- E.g.: lambda phage.
Filamentous phage- E.g.: M13 phage.
Lambda phage vector
Its genome size is about 48,502 bp.
It contains origin of replication, genes for head and tail protein and enzymes for DNA replication
It has more than one recognition sequence for almost all the restriction enzymes.
It should be larger than 38 kb and smaller than 52 kb to packaged into phage particles.
M13 phage vector.
M13 vectors are used to obtain single- stranded copies of cloned DNA.
It is 6407 nucleotides long.
It is circular and 6.4kb in size.
M13 vector only cause infection in F+ and F’ cells.
It is used to produce several copies of M13 mp series of vectors.
Example- M13mp8, M13mp9 etc.
E Artificial chromosome vectors
Artificial chromosomes are circular or linear vectors.
They are stably maintained in, usually 1 to 2 copy per cell.
There are several types of such vectors:
- Bacterial artificial chromosomes (BAC).
- Yeast artificial chromosomes(YAC).
- Mammalian artificial chromosomes(MAC).
- Human artificial chromosome (HAC)
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