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molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word '' cloning'' refers to the fact that the meth ...
, a vector is any particle (e.g.,
plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s, cosmids, Lambda phages) used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed
recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be f ...
. The four major types of vectors are
plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s, viral vectors, cosmids, and
artificial chromosome Artificial chromosome may refer to: * Yeast artificial chromosome * Bacterial artificial chromosome * Human artificial chromosome * P1-derived artificial chromosome A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DN ...
s. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors have an origin of replication, a multicloning site, and a
selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, an ...
. The vector itself generally carries a DNA sequence that consists of an
insert Insert may refer to: *Insert (advertising) *Insert (composites) *Insert (effects processing) *Insert (filmmaking) *Insert key on a computer keyboard, used to switch between insert mode and overtype mode *Insert (molecular biology) *Insert (SQL) *Fi ...
(in this case the
transgene A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change t ...
) 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. All vectors may be used for cloning and are therefore cloning vectors, but there are also vectors designed specially for cloning, while others may be designed specifically for other purposes, such as transcription and protein expression. Vectors designed specifically for the expression of the transgene in the target cell are called
expression vector An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
s, 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. 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 vector A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these ...
s. 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. Insertion of a vector into the target cell is usually called transformation for bacterial cells, transfection for
eukaryotic cells Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...
, although insertion of a viral vector is often called transduction.


Characteristics


Plasmids

Plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s are double-stranded extra chromosomal and generally circular DNA sequences that are capable of replication using the host cell's replication machinery. 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 ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
'', but may also be found in a few eukaryotes, for example in yeast such as ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have b ...
''. 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 A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...
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 vector An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
s, 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,
retrovirus A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptas ...
es leave a characteristic
retroviral integration The pre-integration complex (PIC) is a nucleoprotein complex of viral genetic material and associated viral and host proteins which is capable of inserting a viral genome into a host genome. The PIC forms after uncoating of a viral particle after ...
pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome.


Artificial chromosomes

Artificial chromosomes are manufactured chromosomes in the context of
yeast artificial chromosome Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000  ...
s (YACs),
bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partiti ...
s (BACs), or human artificial chromosomes (HACs). An artificial chromosome can carry a much larger DNA fragment than other vectors. 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.


Transcription

Transcription of the cloned gene is a necessary component of the vector when expression of the gene is required: one gene may be amplified through transcription to generate multiple copies of
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
s, the template on which protein may be produced through translation. A larger number of mRNAs would express a greater amount of protein, and how many copies of mRNA are generated depends on the promoter used in the vector. The expression may be constitutive, meaning that the protein is produced constantly in the background, or it may be inducible whereby the protein is expressed only under certain condition, for example when a chemical inducer is added. These two different types of expression depend on the types of promoter and operator used. 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 Dexamethasone is a glucocorticoid medication used to treat rheumatic problems, a number of skin diseases, severe allergies, asthma, chronic obstructive lung disease, croup, brain swelling, eye pain following eye surgery, superior vena ...
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

Expression vector An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
s produce proteins through the transcription of the vector's insert followed by
translation Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
of the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
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 The Tac-Promoter (abbreviated as Ptac), or tac vector is a synthetically produced DNA promoter, produced from the combination of promoters from the trp and lac operons. It is commonly used for protein production in ''Escherichia coli''. Two hybri ...
, 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
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is ...
s 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 The Kozak consensus sequence (Kozak consensus or Kozak sequence) is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. Regarded as the optimum sequence for initiating translation in ...
: Vectors should encode for a Kozak sequence in the mRNA, which assembles the
ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to fo ...
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. * 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 Ligation may refer to: * Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules * In medicine, the making of a ligature (tie) * Chemical ligation, the production of peptides from amino acids * Tubal ligation, a meth ...
. * Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA. *
Antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention ...
resistance: Vectors with antibiotic-resistance
open reading frame In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible readi ...
s 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. * 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
reporter A journalist is an individual that collects/gathers information in form of text, audio, or pictures, processes them into a news-worthy form, and disseminates it to the public. The act or process mainly done by the journalist is called journalism ...
s 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 A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
* Viral vector * Cloning vector *
Expression vector An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
*
Hybrid vector Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (''in vivo'') or in cell culture (''in vitro''). Viruses have evolved specialized molecula ...
*
Minicircle Minicircles are small (~4 kb) circular replicons. They occur naturally in some eukaryotic organelle genomes. In the mitochondria-derived kinetoplast of trypanosomes, minicircles encode guide RNAs for RNA editing. In ''Amphidinium'', the chlorop ...
*
Recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be f ...
* Naked DNA * Vector (epidemiology), an organism that transmits disease * Human artificial chromosomes *
Yeast artificial chromosome Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000  ...
s *
Bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partiti ...
s * DNA vaccination


References


Further reading

*


External links


Waksman Scholars introduction to vectors
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