VECTOR

INTRODUCTION TO VECTOR

DEFINITION

                        ‘’A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.’’
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.
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
Purpose of a 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. 
To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a synthetic multiple cloning site (MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic resistance [e.g. beta-lactamase (see figure)] is often carried by the vector to allow the selection of positively transformed cells (see Screening below). All plasmids must carry a functional origin of replication (ORI; not shown in figure).
The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes.
Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker

Origin of replication

 The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses. DNA replication may proceed from this point bidirectionally or unidirectionally.

The specific structure of the origin of replication varies somewhat from species to species, but all share some common characteristics such as high AT content. The origin of replication binds the pre-replication complex, a protein complex that recognizes, unwinds, and begins to copy DNA.

Types

The two types of replication origin are :

• Narrow or broad host range
• High- or low-copy number

There are also significant differences between prokaryotic and eukaryotic origins of replication:

• Bacteria have a single circular molecule of DNA, and typically only a single origin of replication per circular chromosome.
• Archaea have a single circular molecule of DNA, and several origins of replication along this circular chromosome.
• Eukaryotes often have multiple origins of replication on each linear chromosome that initiate at different times (replication timing), with up to 100,000 present in a single human cell.Having many origins of replication helps to speed the duplication of their (usually) much larger store of genetic material. The segment of DNA that is copied starting from each unique replication origin is called a replicon.

Multiple cloning site

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids.Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology, bioengineering, and molecular genetics, MCSs let a biotechnologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs).

EXAMPLE

 The pUC18 and pUC19 polylinker

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, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell.
Selectable markers are often antibiotic resistance genes; bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material.
Examples of selectable markers include:the Abicr gene

Common vectors

Two common vectors are plasmids and viral vectors.

Plamid

A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.. They are double-stranded and, in many cases, circular.
Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).Plasmid sizes vary from 1 to over 1,000 kilobase pairs (kbp). The number of identical plasmids in a single cell can range anywhere from one to even thousands under some circumstances.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.
Illustration of a bacterium with plasmid enclosed showing chromosomal DNA and plasmid
Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host.
Plasmids can be found in all three major domains: Archea, Bacteria and Eukarya.
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 host gene expression allowing the intentional uptake of the genetic element by transformation.
 Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage when nutrients are scarce.

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episomes, the lower example, integrate into the host chromosome.

However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.

CONSTRUCTION OF  PLASMID VECTOR.

Plasmid can be good cloning vector because they carry an origin of replication and are therefore able to replicate independently within cell.

Most plasmid used as vectors also encode some type of selectable marker such as gene of resistance as amphiciline.

If  the host cell are amphiciline sensitive,the only host cell can grow on medium containing amphiciline are thosethat have taken up plasmid.

Vector must also havue a small sequence of base pair that can be recognized by restriction enzyme.when this enzyme open the circular plasmid ,foreign DNA can be incorporated.

When plasmid vector and foreign DNA are both cut with same restriction enzyme and mixed together, not all molecule will join to form recombinant.

Some vector molecule will reaneal without incorporating foreign DNA .To identify cells that contain plasmids have incorporated foreign DNA a second marker gene is needed on the vector.

Episomes

An episome is a portion of genetic material that can exist independent of the main body of genetic material (called the chromosome) at some times, while at other times is able to integrate into the chromosome.Examples of episomes include insertion sequences and transposons.
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 (transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease).

Yeast plasmids

Other types of plasmids are often related to yeast cloning vectors that include:

• Yeast integrative plasmid (YIp), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C);
• Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can "get lost" during the budding.

 Viral vector
Viral vectors are a tool 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 molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture.

Key properties of a viral vector

Viral vectors are tailored to their specific applications but generally share a few key properties.

• Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions.
• Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects.
• Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design.
• Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral

 Types of viral vectors

Retroviruses

 Retroviruses are one of the mainstays of current gene therapy approaches. The recombinant retroviruses such as the Moloney murine leukemia virus have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase that allows integration into the host genome..

The primary drawback to use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for transduction. As a result, cells such as neurons are very resistant to infection and transduction by retroviruses. There is concern that insertional mutagenesis due to integration into the host genome might lead to cancer or leukemia.
Lentiviruses
Lentiviruses are a subclass of Retroviruses. They have recently been adapted as gene delivery vehicles (vectors) thanks to their ability to integrate into the genome of non-dividing cells, which is the unique feature of Lentiviruses as other Retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides.

Adenoviruses

Adenoviral DNA does not integrate into the genome and is not replicated during cell division. This limits their use in basic research, although adenoviral vectors are occasionally used in in vitro experiments. Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences. To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.

Adeno-associated viruses

Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy.[1]

Nanoengineered substances

Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate.)

Expression vector and its construction

An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin that are important for medical treatments of specific diseases like diabetes.
Expression vectors are used for molecular biology techniques such as site-directed mutagenesis. Cloning vectors, which are very similar to expression vectors, involve the same process of introducing a new gene into a plasmid, but the plasmid is then added into bacteria for replication purposes. In general, DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein.

EXPRESSION VECTOR.

Shuttle vector

 A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species [1]. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli then used in a system which is more difficult or slower to use (e.g. yeast, other bacteria).

Shuttle vectors include plasmids that can propagate in eukaryotes and prokaryotes (e.g. both Saccharomyces cerevisiae and Escherichia coli) or in different species of bacteria (e.g. both E. coli and Rhodococcus erythropolis). There are also adenovirus shuttle vectors, which can propagate in E. coli and mammals.Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli (amplification). They can also be used for in vitro experiments and modifications (e.g. mutagenesis, PCR)