Features
Modern vectors contain essential components as well as other additional features:
- Origin of replication: Necessary for the replication and maintenance of the vector in the host cell.
- Promoter: Promoters are used to drive the transcription of the vector's transgene as well as the other genes in the vector such as the antibiotic resistance gene. Some cloning vectors need not have a promoter for the cloned insert but it is an essential component of expression vectors so that the cloned product may be expressed.
- Cloning site: This may be a multiple cloning site or other features that allow for the insertion of foreign DNA into the vector.
- Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.
- Antibiotic resistance: Vectors with antibiotic-resistance open reading frames allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection.
- Epitope: Vector contains a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the target protein.
- Reporter genes: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-α which codes for the N-terminus fragment of β-galactosidase, an enzyme that digests galactose. A multiple cloning site is located within lacZ-α, and an insert successfully ligated into the vector will disrupt the gene sequence, resulting in an inactive β-galactosidase. Cells containing vector with an insert may be identified using blue/white selection by growing cells in media containing an analogue of galactose (X-gal). Cells expressing β-galactosidase (therefore doesn't contain an insert) appear as blue colonies. White colonies would be selected as those that may contain an insert. Other commonly used reporters include green fluorescent protein and luciferase.
- Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria.
- Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein. The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag allows the tag to be removed later.
Read more about this topic: Vector (molecular Biology)
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