Cloning vectors

Image 1: Cloning vectors

The concept of cloning vectors is similar to the example described below. Imagine you want to reach a destination far away from your house. So, you use a vehicle to reach that place, isn’t it? The vehicle carries you to the desired destination. In the molecular world, the extrachromosomal DNA known as the plasmids act as vehicles in carrying the fragment of interest to the desired location. Thus, in DNA cloning, a plasmid or phage chromosome acts as a vector (vehicle) to carry the cloned DNA segment. There are many types of vectors such as plasmids, cosmids, phagemids, shuttle vectors, expression vectors, and artificial chromosomes.

1.   Plasmid vectors:

The extrachromosomal elements present in the bacterial cells replicate autonomously or independently. Plasmids have a circular DNA with double strands. It consists of sequences capable of independent function. Naturally prevalent plasmids serve as the main source of plasmid vectors. They are engineered to get the desired features. Plasmid extraction is easily possible with the E. coli bacterial cells. These plasmids pass from one cell to another cell. Replication of plasmids occurs during each cell division. We obtain plasmids through disruption of the bacterial cells followed by centrifugation. Next step involves treatment with the restriction enzymes. Cleavage of a plasmid produces staggered ends. The foreign DNA gets incorporated in the vacant site. Note the two important points about the plasmids. Firstly, the plasmids possess a limited number of restriction sites. Secondly, the plasmids carry genes for resistance to antibiotics. Care must be taken to avoid incorporation of any harmful gene into it. 

Image 2: Plasmid Vector

The features of E. coli plasmids are as follows:

·        Origin of replication: The site for starting or initiating the replication is known as the origin of replication. It permits an independent replication of the plasmid.

·        Selectable marker: It is usually dominant. A selectable marker gets readily detected. Thus, it exhibits a trait suitable for artificial selection. One more type of marker is known as a screenable marker. It allows the researchers to distinguish between the required cells and the unwanted cells. An antibiotic resistance gene acts as a selectable marker. Examples include ampicillin or a tetracycline resistance gene. The positive and negative markers are the two types of markers. The markers conferring a selective advantage to the host are known as positive markers. The markers eliminating the growth of the markers are known as negative markers.

·        Restriction enzyme cleavage sites: The restriction sites are present just once in a vector. The DNA fragments get inserted into the vector at specific sites. An appropriate restriction enzyme recognizing this sequence gets selected for cleaving that particular site. Later on, the fragment of interest gets inserted into that site. Some of the plasmids have multiple cloning sites. Plasmids act as disease models. Protein productions and gene therapy also involve the use of plasmid vectors.

Example of a plasmid vector: pUC 19 vector

pUC 19 or “puck” 19 vector is a 2686 base pair vector with multiple cloning sites. They are unique restriction sites clustered in one region. A high copy number of the plasmids and amp^R selectable marker make it a good choice for cloning experiments. The polylinker has restriction sites for various restriction enzymes such as Eco RI, Bam HI, HindIII, PstI, SacI, Sma I and many other enzymes. The polylinker is within the beta-galactosidase gene (lac z+). The pUC 19 vector gets cleaved at its polylinker site leaving a vacant space. Next, a piece of DNA to be cloned gets separated through electrophoresis and obtained for further procedures. The piece of DNA, the vector, and the ligating enzymes are mixed. The DNA gets inserted into the plasmid and the gaps sealed by DNA ligase. The resulting plasmid gets transformed into E. coli. It involves incubation of the recombinant plasmids with E. coli cells and treating chemically or by electroporation. Next step includes plating of the transformed cells on a suitable medium to observe the colonies. Blue-white screening distinguishes recombinant DNA clones from the plasmids. Episomes are the plasmids capable of integrating into the chromosome.

2.   Phagemids or Phasmid:

It is a cloning vector with two properties such as bacteriophage and plasmid properties. They are DNA based vectors. These phasmids exhibit a property of getting packed into phage heads.

3.   Fosmids:

They resemble cosmids and involve bacterial F plasmids. E. coli serves as a source of fosmid vectors and contains only one fosmid.

4.   Shuttle vectors:

The vectors introduced into two or more organisms are known as shuttle vectors. For example, some vectors get transformed into E. coli and also get transformed into the yeast. These plasmids propagate in the prokaryotes and eukaryotes. Example, an Adenovirus shuttle vector propagates into both prokaryotes and mammals. Multiple copies of a gene get synthesized. A shuttle vector consists of two origins of replication specific to each host. Shuttle vectors are also known as bifunctional vectors. A yeast shuttle vector consists of bacterial origin of replication, yeast origin of replication, yeast selectable marker, bacterial selectable markers, and an origin of transfer.

5.   Cosmids:

Cosmids are also known as hybrid plasmids. They consist of a lambda phage cos sequence. The genetic engineering technique uses cosmids as cloning vectors. They have a suitable origin of replication and an antibiotic resistance gene. These plasmids get packaged into phage capsids thereby allowing transduction. The cos sequences present in a cosmid are just a few hundred base pairs in length. The availability of the cosmids as the shuttle or mammalian cosmids depends on the need of the experiment. A typical feature of the cosmid involves the formation of colonies instead of forming plaques.

6.   Expression vectors:

A plasmid or a virus behaves like an expression vector. However, an expression vector designing must boost the expression of the desired gene. The gene of interest gets inserted into the expression vector. The gene contains a protein-coding region. The expression of the desired gene produces a protein product.

Image 3: The Expression vector

7.   Plant vectors:

The plant vectors involve Ti-plasmid vectors. The Ti-plasmids belong to a soil bacterium known as Agrobacterium tumefaciens. It consists of T-DNA capable of integrating into a plant genome. Integration of plasmid into the plant genome occurs when the bacterium infects the plant and causes crown gall disease. An example of plant cloning vector involves the pBIN vector. It consists of a lac z’ gene, a right T-DNA boundary, a left T-DNA boundary, an origin of replication, and kanamycin resistance gene.

8.   BAC’s and YAC’s:

The bacteria exchange their plasmids through a process known as conjugation. The donor bacterial cell transfers its F plasmid through a cellular bridge. BACs or bacterial artificial chromosome construction involves F-plasmids. The human genome project utilized bacterial artificial chromosomes for various projects.

·        Components of BACs:

·        oriS, rep E-F: It is the origin of replication. It regulates the copy number of the plasmids.

·        The parA and par B components help in the partition of the cells into daughter cells during cell division.

·        Antibiotic resistance gene acts as a selectable marker.

·        Phage promoters induce transcription of an inserted gene such as T1 and Sp6 promoters.

Various disease studies involve BACs as models. A BAC library is a collection of BAC clones stored in regulated temperatures. Several genes and large gene studies involve BACs. They are useful in the development of vaccines.

Yeast artificial chromosomes or YACs help in cloning large DNA thereby guiding in an accurate construction of a physical map. They involve genetically engineered yeast DNA.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

[3] Gene Cloning and Manipulation, Christopher Howe
[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

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