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Vectors

01/09/2013 14:01

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.

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 (transgene) 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 cloning vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Cloning 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.

Features of a good vector

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.

· high copy number (number of plasmids maintained per 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 (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-ransferase, 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.

Plasmid

A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small circular, double-stranded DNA molecules in bacteria, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids carry genes that may benefit survival of the organism (e.g. antibiotic resistance), and can frequently be transmitted from one bacterium to another (even of another species) via horizontal gene transfer. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms.

Plasmid sizes vary from 1 to over 1,000 kbp.

The number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances.

Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.

The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.

Plasmids are considered replicons, capable of replicating autonomously within a suitable host.

Plasmids can be found in all three major domains:Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life.

Unlike viruses, plasmids are naked DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer.

Plasmid host-to-host transfer requires direct mechanical transfer by conjugation, or changes in incipient host gene expression allowing the intentional uptake of the genetic element by transformation.

Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a detrimental or commensal state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.

Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances. Plasmids can also provide bacteria with the ability to fix nitrogen or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.

Plasmids as vectors

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.Many plasmids are commercially available for such uses.

The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.

Next, the plasmids are inserted into bacteria by a process called transformation.

Then, the bacteria are exposed to the particular antibiotics.

Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter to select only the modified bacteria.

Now these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example,insulin or even antibiotics.

A plasmid can contain inserts of up to 30-40 kbp.

Applications

Disease models:

Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid-based techniques precluded their use in the creation of more accurate human cell models. However, developments in Adeno-associated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.

Gene therapy:

Some strategies of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double-strand break to the DNA genome and cause homologous recombination. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage, cancer-causing mutations, or an immune response is avoided.

Types

One way of grouping plasmids is by their ability to transfer to other bacteria.

Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium.

Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids.

An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell.

Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Thus, plasmids can be assigned into incompatibility groups.

Another way to classify plasmids is by function. There are five main classes:

· Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.

· Resistance (R)plasmids, which contain genes that provide resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.

· Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.

· Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid.

· Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups.

Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems that attempt to actively distribute a copy to both daughter cells. These systems, which include the parABS system and parMRC system, are often referred to as the partition system or partition function of a plasmid.

Conformations

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

· Nicked open-circular DNA has one strand cut.

· Relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself.

· Linear DNA has free ends, either because both strands have been cut or because the DNA was linear in vivo. This can be modeled with an electrical extension cord that is not plugged into itself.

· Supercoiled (or covalently closed-circular) DNA is fully intact with both strands uncut, and with an integral twist, resulting in a compact form. This can be modeled by twisting an extension cord and then plugging it into itself.

· Supercoiled denatured DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

pBR322

The first plasmid vector that has been constructed artificially is pBR322.

It is named after the scientists Bolivar and Rodriguiz who constructed it in 1977.

It is 4362bp in size.

It has an origin of replication derived from a colicin-resistance plasmid (ColE1).

This origin allows a fairly high copy number, about 100 copies of the plasmid per cell.

Plasmid pBR322 carries two selectable markers viz. genes for resistance to ampicillin (Apr) and tetracycline (Tcr ).

Several unique RE sites are present within these genes for insertion of foreign DNA. When a foreign DNA segment is inserted in any of these genes, the antibiotic resistance by that particular gene is lost. This is called insertional inactivation. For instance, insertion of a restriction fragment in the SalI site of the Tcr gene inactivates that gene. One can still select for Apr colonies, and then screen to see which ones have lost Tcr .

pUC

A series of small plasmids (about 2.7 kb) have been developed at the University of California and hence the name pUC e.g. pUC7, 8, 18 and 19 etc.

These are high copy number plasmids that carry an ampicillin resistance gene and an origin of replication, both from pBR322.

They also have a multiple cloning site (MCS) – a sequence of DNA that carries unique sites for many REs.

The MCS contains a portion of lacZ gene that codes for the enzyme β-galactosidase. When such plasmids are introduced into E. coli, the colonies are blue on plates containing X-gal (substrate for β- galactosidase) and IPTG (isopropyl thiogalactoside, an inducer). When a foreign DNA is introduced in MCS, the β-galactosidase activity is lost. Thus cells containing recombinant plasmids form white (not blue) colonies.

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.

Bacteriophage

Basic features of bacteriophages Bacteriophages, or phages as they are commonly known, are viruses that specifically infect bacteria.

Like a11 viruses, phages are very simple in structure, consisting merely of a DNA (or occasionally ribonucleic acid (RNA)) molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules.

Lytic cycle

The general pattern of infection, which is the same for all types of phage, is a three-step process:

(1) The phage particle attaches to the outside of the bacterium and injects its DNA chromosome into the cell.

(2) The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on the phage chromosome.

(3) Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium.

With some phage types the entire infection cycle is completed very quickly, possibly in less than 20min. This type of rapid infection is called a lytic cycle, as release of the new phage particles is associated with lysis of the bacterial celL The characteristic feature of a lytic infection cycle is that phage DNA replication is immediately followed by synthesis of capsid proteins, and the phage DNA molecule is never maintained in a stable condition in the host cell.

Lysogenic phages

In contrast to a lytic cycle, lysogenic infection is characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions. With many lysogenic phages the phage DNA is inserted into the bacterial genome, in a manner similar to episomal insertion. The integrated form of the phage DNA (called the prophage) is quiescent, and

a bacterium (referred to as a lysogen) that carries a prophage is usually physiologically indistinguishable from an uninfected cell. However, the prophage is eventually released from the host genome and the phage reverts to the lytic mode and lyses the cell. a typical lysogenic

phage of this type,is the lambda phage.

A limited number of lysogenic phages follow a rather different infection cycle. When M13 or a related phage infects E. coli, new phage particles are continuously assembled and released from the cell. The M13 DNA is not integrated into the bacterial genome and does not become quiescent. With thesephages, cell lysis never occurs, and the infected bacterium can continue to grow and divide, albeit at a slower rate than uninfected cells.

Phage vectors can accommodate more DNA (upto 25 kb) than plasmids and are often used for preparation of genomic libraries.

They also have higher transformation efficiency as compared to plasmids.

Two bacteriophages namely, Lambda (λ) and M13 have been commonly used for construction of vectors for cloning in E. coli.

Lambda (λ) phage vectors

Lambda is a temperate bacteriophage with a genome size of 48.5 kb. Its entire DNA sequence is known.

The lambda genome is a linear, double-stranded molecule with single-stranded, complementary ends.

These ends can hybridize with each other (and do so when the DNA is within an infected cell) and are thus termed cohesive (cos) sites.

The phage can have two modes of life cycles i.e. lytic and lysogenic.->(click here for animation)

During lytic cycle, it replicates independently in the host cell and produces a large number of phage particles which are released by lysis of the host. Alternatively, it can take up lysogenic growth, meaning that it integrates its DNA into the bacterial chromosome and multiplies along with it.

Two types of vectors have been constructed from lambda phage.

Theses are insertional and replacement vectors.

Insertional vectors have one unique restriction site for a particular restriction enzyme and can accommodate 6-7 kb DNA.

Examples of insertional vectors are λgt10, λgt11 and λZAP II.

On the other hand, replacement vectors have two cleavage sites for a restriction enzyme and can accommodate up to 25 kb DNA.

When vector is cut with a restriction endonuclease, a stuffer fragment is removed and replaced with a foreign DNA. Some examples of replacement vectors are EMBL3, EMBL3A, EMBL4, λDASH, λFIX, GEM11 and GEM12.

Bacteriophage lambda can be reconstituted in a test tube by simply mixing phage DNA with a mixture of phage proteins, an infective viral particle with the DNA inside the phage head can be produced. This process is called in vitro packaging. There is a strict size requirement for the piece of DNA that goes into the phage head. That is, it should not be more than 52 kb and less than 38 kb. This feature allows only the recombinants to be packaged inside the phage head.

In addition, some lambda phage vectors have a stuffer fragment that carries the β-galactosidase gene. When it is removed or when foreign DNA is cloned within the gene, β-galactosidase activity may be abolished. The accompanying loss of activity may be used to select recombinant clones.

M13 Phage vectors

M13 is a filamentous bacteriophage of E. coli and contains a single stranded circular DNA of 7.2 kb.

M13 life cycle