In molecular cloning, a vector is a DNA molecule used as a vehicle to
artificially carry foreign genetic material into another cell, where it can
be replicated and/or expressed. A vector containing foreign DNA is termed
recombinant DNA. The four major types of vectors are plasmids, viral vectors,
cosmids, and artificial chromosomes. Of these, the most commonly used vectors
are plasmids. Common to all engineered vectors are an origin of replication, a
multicloning site, and a selectable marker.
The vector itself is generally a DNA sequence that consists of an insert and
a larger sequence that serves as the "backbone" of the vector. The purpose of
a vector which transfers genetic information to another cell is typically
to isolate, multiply, or express the insert in the target cell. Vectors
called expression vectors specifically are for the expression of the transgene
in the target cell, and generally have a promoter sequence that drives expression
of the transgene. Simpler vectors called transcription vectors are only capable
of being transcribed but not translated: they can be replicated in a target cell
but not expressed, unlike expression vectors. Transcription vectors are used
to amplify their insert. Insertion of a vector into the target
cell is usually called transformation for bacterial cells, transfection for
eukaryotic cells, although insertion of a viral vector is often called
transduction. Characteristics
= Plasmids = Plasmids are double-stranded and
generally circular DNA sequences that are capable of automatically replicating
in a host cell. 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. 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 cloning purposes. These plasmid are generally non-conjugative but may have
many more features, notably a "multiple cloning site" where multiple restriction
enzyme cleavage sites allow for the insertion of a transgene insert. The
bacteria containing the plasmids can generate millions of copies of the
vector within the bacteria in hours, and the amplified vectors can be extracted
from the bacteria for further manipulation. Plasmids may be used
specifically as transcription vectors and such plasmids may lack crucial
sequences for protein expression. Plasmids used for protein expression,
called expression vectors, would include elements for translation of protein,
such as a ribosome binding site, start and stop codons.
= Viral vectors = Viral vectors are generally genetically
engineered viruses carrying modified viral DNA or RNA that has been rendered
noninfectious, but still contain viral promoters and also the transgene, thus
allowing for translation of the transgene through a viral promoter.
However, because viral vectors frequently are lacking infectious
sequences, they require helper viruses or packaging lines for large-scale
transfection. Viral vectors are often designed for permanent incorporation of
the insert into the host genome, and thus leave distinct genetic markers in
the host genome after incorporating the transgene. For example, retroviruses
leave a characteristic retroviral integration pattern after insertion that
is detectable and indicates that the viral vector has incorporated into the
host genome. Transcription
Transcription is a necessary component in all vectors: the premise of a vector
is to multiply the insert. Thus, even stable expression is determined by
stable transcription, which generally depends on promoters in the vector.
However, expression vectors have a variety of expression patterns:
constitutive or inducible. This expression is based on different
promoter activities, not post-transcriptional activities. Thus,
these two different types of expression vectors depend on different types of
promoters. 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.
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
Drosophilia heat shock promoter only initiates after high temperatures.
Expression Expression vectors produce proteins
through the transcription of the vector's insert followed by translation
of the mRNA produced, they therefore require more components than the simpler
transcription-only vectors. Expression in different host organism would require
different elements, although they share similar requirements, for example a
promoter for initiation of transcription, a ribosomal binding site
for translation initiation, and termination signals.
= 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 a hybrid of both the
Trp and Lac Operon promoters. Ribosome binding site 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 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 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: 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. Cells expressing
β-galactosidase 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.
See also Plasmid
Viral vector Cloning vector
Expression vector Minicircle
Recombinant DNA Naked DNA
Vector, an organism that transmits disease
References Freshney, Ian R. Culture of Animal
Cells: A manual of basic technique. John Wiley & Sons, Inc., Hoboken, New Jersey.
ISBN 978-0-471-45329-1 External links
Waksman Scholars introduction to vectors A comparison of vectors in use for
clinical gene transfer Gene Transport Unit