Vector (molecular biology)

In

The manipulation of DNA is normally conducted on E. coli vectors, which contain elements necessary for their maintenance in E. coli. However, vectors may also have elements that allow them to be maintained in another organism such as yeast, plant or mammalian cells, and these vectors are called shuttle vectors. Such vectors have bacterial or viral elements which may be transferred to the non-bacterial host organism, however other vectors termed intragenic vectors have also been developed to avoid the transfer of any genetic material from an alien species.^[3]

Insertion of a vector into the target cell is usually called

Plasmids are double-stranded extra chromosomal and generally circular DNA sequences that are capable of replication using the host cell's replication machinery.^[7] Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host. Plasmids are found widely in many bacteria, for example in Escherichia coli, but may also be found in a few eukaryotes, for example in yeast such as Saccharomyces cerevisiae.^[8] Bacterial plasmids may be conjugative/transmissible and non-conjugative:

• conjugative - mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids.
• nonconjugative - do not mediate DNA through conjugation, e.g., many R and col plasmids.

Plasmids with specially-constructed features are commonly used in laboratory for

Artificial chromosomes

Artificial chromosomes are manufactured chromosomes in the context of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or human artificial chromosomes (HACs). An artificial chromosome can carry a much larger DNA fragment than other vectors.^[9] YACs and BACs can carry a DNA fragment up to 300,000 nucleotides long. Three structural necessities of an artificial chromosome include an origin of replication, a centromere, and telomeric end sequences.^[10]

Transcription

Viral promoters are often used for constitutive expression in plasmids and in viral vectors because they normally force constant transcription in many cell lines and types reliably.^[13] Inducible expression depends on promoters that respond to the induction conditions: for example, the murine mammary tumor virus promoter only initiates transcription after dexamethasone application and the Drosophila heat shock promoter only initiates after high temperatures.

Some vectors are designed for transcription only, for example for in vitro mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.

Expression

Prokaryotes expression vector

• Promoter - commonly used inducible promoters are promoters derived from lac operon and the T7 promoter. Other strong promoters used include Trp promoter and Tac-Promoter, which are a hybrid of both the Trp and Lac Operon promoters.
• Ribosome binding site
  (RBS) - follows the promoter, and promotes efficient translation of the protein of interest.
• Translation initiation site -
  Shine-Dalgarno sequence
  enclosed in the RBS, 8 base-pairs upstream of the AUG start codon.

Eukaryotes expression vector

Eukaryote expression vectors require sequences that encode for:

• Polyadenylation tail: Creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA production.
• Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors.
• Kozak sequence: Vectors should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA.

Features

Modern artificially-constructed vectors contain essential components found in all vectors, and may contain other additional features found only in some vectors:

• Origin of replication: Necessary for the replication and maintenance of the vector in the host cell.
• 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 through ligation.
• 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: Some vectors may contain a sequence for a specific epitope that can be incorporated into the expressed protein. It allows for antibody identification of cells expressing the target protein.
• blue/white selection by growing cells in media containing an analogue of galactose (X-gal). Cells expressing β-galactosidase (therefore does not 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.
• 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.

• Plasmid
• Viral vector
• Cloning vector
• Expression vector
• Hybrid vector
• Minicircle
• Recombinant DNA
• Naked DNA
• Vector (epidemiology)
  , an organism that transmits disease
• Human artificial chromosomes
• Yeast artificial chromosomes
• Bacterial artificial chromosomes
• DNA vaccination

References