Salient features of a cloning vector

In the past few decades, the field of molecular biology has made some fascinating discoveries and the majority of them used plasmid vectors as a pivotal tool which allowed them to explore novel elements for molecular features of life. Similarly, vectors are also used worldwide for biotechnological advances, for example, insulin production using a strain of E.coli to treat diabetes and many others. The cloning vector is an important component of genetic engineering. So let us study different cloning vectors and their features.

What is a cloning vector?

To begin with, cloning vectors are used generally in molecular cloning, a vector is a foreign DNA molecule used as a carrier to artificially carry foreign genetic material into the host body, where it multiplies and sometimes can be expressed.

The cloning vectors have limited size for inserting genetic material that they carry and transfer, depending on the size and the application one must choose the suitable vector for the desired purpose.

Moreover, different types of vectors are available for cloning such as plasmids, bacteriophages, bacterial artificial chromosomes (BACs), mammalian artificial chromosomes (MACs), etc.

Characteristics of a cloning vector

The cloning vector as a carrier molecule should have a few features in general such as:

1. The vector should have an ori site so that it can self-replicate.
2. It must have a unique restriction site for targeted DNA.
3. It should have a selectable marker
4. The vector should be easily isolated from the host cell.
5. It should have multiple cloning sites

Types of cloning vector

• Plasmid

Plasmids are the first vectors used in gene cloning, plasmids are generally found in bacteria, and eukaryotes. They are extrachromosomal, autonomous replicating DNA molecules. They are particularly inserted into bacterial cells by a process known as transformation. Moreover, they have antibiotic resistance genes and have a decent copy number.

Some examples: pBR322, F-plasmid, col plasmid

• Bacteriophage

Bacteriophage turns out to be more efficient vectors than plasmids as they can carry large DNA inserts for cloning. These are an intracellular obligate parasite, that multiplies using some of the host enzymes.

Example : M13, Phage lambda.

• Phagemids

Phagemids are artificial vectors used in combination with M13 bacteriophages. Phagemids have multiple cloning sites and an inducible lac gene promoter. Blue-white screening is used to identify phagemids.

For instance: pComb3XSS

Image Source: Levisson, M., Spruijt, R. B., Winkel, I. N., Kengen, S. W., & Oost, J. V. D. (2014). Phage display of engineered binding proteins. In Protein Downstream Processing (pp. 211-229). Humana Press, Totowa, NJ.

• Bacterial artificial chromosomes

BACs are simple plasmids designed to clone very large DNA fragments, similar to E.coli plasmid vectors. Moreover, BACs are generally used in genome projects to study genetic disorders.

Image source: Hain, T., Otten, S., von Both, U., Chatterjee, S. S., Technow, U., Billion, A., … & Chakraborty, T. (2008). Novel bacterial artificial chromosome vector pUvBBAC for use in studies of the functional genomics of Listeria spp. Applied and environmental microbiology, 74(6), 1892-1901.

• Human artificial chromosome

Also known as mammalian artificial chromosomes, HACs are still under development and can be used as vectors in the transfer of new genes and the study of their expression and chromosomal function.

Features required by cloning vector

• Ori

Origin of replication from the name itself one can understand, a sequence from where replication starts, pieces of DNA linked to the ori can replicate and make multiple copies within the host cell. Secondly, the ori is also responsible for a number of copies made of the DNA molecule. So if one wants to have many copies of the target DNA then the vector chosen for cloning should support high copy numbers.

Also, read- Biotechnology Archives – My Biology Dictionary

• Selectable markers

Selectable markers play an important role in vector as it helps in identifying and eliminating non-transformants and selectively permitting only the growth of transformants. Transformation is a procedure to introduce a DNA piece into a host.

• Cloning sites

Cloning sites are the unique recognition sites of restriction enzymes. Particularly, at the restriction site present in one of the two antibiotic resistance genes, the ligation of alien DNA is carried out.

Cloning into one of these sites inactivates the gene allowing recombination to be differentiated from non-recombinants known as insertional inactivation.

• Vectors for cloning genes in plants and animals

For plant  – Pathogenic vectors like Agrobacterium tumefaciens, a pathogen for several dicot plants can cause a change in plant cells to the tumor and direct them to produce chemicals required by the pathogen.

Now tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens can be modified into a cloning vector and is used to deliver the desired genes to a variety of plants.

For animals–  Similarly for humans, retrovirus which transforms normal cells into cancerous cells now can be disarmed and used as a cloning vector to deliver genes of interest into different animal cells. For instance, other examples are Adenovirus, papillomavirus in mammals, tobacco mosaic virus Gemini viruses in plants.

Image source: www.molecularcloud.org

To summarize, biotechnological processes are incomplete without cloning vectors.

Also, read- plasmid / plasmids | Learn Science at Scitable (nature.com)