Plasmid Vectors For Gene Cloning
We all know, various tools are needed for genetic engineering out of which vectors serve as the major tools for gene cloning inside the host cell. Genetic transformations take place with the help of key tools in biotechnology. The tools include restriction enzymes, polymerase enzymes, ligases, vectors, and the host organism. Vectors are one such tool that acts as a vehicle for the transfer of foreign genetic material into another cell. Some of the commonly used cloning vectors are PLASMIDS and BACTERIOPHAGES. Plasmid vectors for gene cloning are well-known. They have the power of replicating inside the host cell thus making multiple copies of it.
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What are Plasmids?
Plasmids are extra-chromosomal circular DNA sequences. They are capable of automatically replicating in a host cell. They are not a part of the nuclear content of the cell and are generally double-stranded DNA. These are found in bacterial cells. One special characteristic of plasmids is that it replicates independently of the control of chromosomal DNA. Almost all plasmids consist of a transgene insert and an origin of replication, allowing semi-independent replication of the plasmid in the host. It allows for semi-independent replication of the plasmid in the host. Plasmids almost always carry one or more genes along with them. These are often responsible for a useful characteristic displayed by the host bacterium.
Plasmids stand to fit the criteria to be able to act as a vector for gene cloning. Firstly and most importantly it is able to replicate inside the host cell such that the genetic material can be passed on to the daughter cells. Secondly, it is small in size (nearly 10 kb in) otherwise it would break down during purification and is also difficult to manipulate.
Plasmids Vectors For Gene Cloning
Vectors used presently, are engineered in such a way that helps in the easy linking of foreign DNA, and the selection of recombinants from non-recombinants. Plasmids can be conjugative/transmissible or non-conjugative. Conjugative plasmids mediate DNA transfer through conjugation and spread rapidly among a population’s bacterial cells. Nonconjugative plasmids do not mediate DNA through conjugation. They are generally written with the p prefix. Example: pbr322, pGEX-3X.
Size And Copy Number Of Plasmid vectors for gene cloning
One of the most pre-requisite qualities of a cloning vector is to have a large copy number and a relevantly small size. The size and copy number of a plasmid vector is important as far as cloning is considered. As mentioned, a size of less than 10 kb is most desirable for a cloning vector. The plasmids range from 1 kb to 250 kb in size out of which very few are compatible with cloning purposes. The higher the copy number, the better the vector is considered. The copy number refers to the number of molecules of an individual plasmid that are normally found in bacterial cells. Being to the point, multiple copies of the cloning vector in a cell are more favourable so that large quantities of recombinant DNA molecules can be obtained.
Conjugation And Compatibility Of Plasmid vectors for gene cloning
As mentioned earlier plasmids fall into two categories, conjugative and non-conjugative. Conjugative plasmids have the ability for sexual conjugation between bacterial cells. Here, conjugation refers to the transfer of a set of genes from the parent to the daughter bacterium. Conjugation is controlled by the transfer genes (tra gene) which are present in conjugating plasmids but absent in non-conjugative types. More than one type of conjugation plasmids can be present in a single cell at a time. For example, in E.coli. To be able to co-exist in the same cell, these plasmids must be compatible while the non-compatible ones are those that will be rapidly lost from the cell. This defines the in-compatibility group on basis of whether or not they can co-exist.
Plasmids In Organisms Other Than Bacteria
As we all know bacterias are the ones that solely has plasmid as an organelle and by no means it is common in other
organisms. However, the best characterized eukaryotic plasmid is the 2 Fm circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The search for plasmids in eukaryotic cells has been proven meaningless because higher organisms simply do not harbour plasmids in their cells.
Features Of Plasmid Vectors For Gene Cloning
ORIGIN OF REPLICATION (ORI)
This is the part of the plasmid vector from where replication starts. Any piece of DNA linked to the ori sequence can be made to replicate within the host. Therefore a large number of copies or clones of the target DNA can be attained with a vector that supports high copy numbers.
A plasmid vector requires a feature that could help distinguish the recombinants. It helps eliminate the non-recombinants and selectively permits the growth of recombinants. Genes coding for resistance to antibiotics are commonly used as selectable markers. These could be the ones encoding resistance to ampicillin, tetracycline, chloramphenicol, etc. The transformed cells would have resistance to those antibiotics and grow in their presence, while the non-recombinants will not thrive.
RECOGNITION OR CLONING SITES
The vector must contain perhaps a recognition site for the commonly used restriction enzyme so that the foreign DNA could be linked to it. Restriction endonuclease enzymes are the ones that cut the DNA (here the plasmid) at specific palindromic sequences. Thus the vector will require a specific recognition point for that restriction enzyme to identify the point of linkage of the alien DNA. Preferably there should be only one particular recognition site for that restriction enzyme. The presence of more than one recognition site will generate a lot of fragments and complicate the cloning process.
Applications of Plasmid Vectors For Gene Cloning
- Plasmids have a wide range of applications in the field of biotechnology. Apart from cloning, there’s also PCR and sequencing where it is useful.
- Having multiple cloning sites makes the plasmid an easy-to-modify vector.
- They are easier to get sequenced by the foreign DNA with the help of restriction enzymes.
- Recently plasmids have been experimented with to transfer functional genes to potentially deficient ones. Thus being looked forward to as an alternative to gene therapy.
Plasmid vectors are being widely used in the genetic engineering and biotechnology. With the advancement in this fields plasmids have provided their worth as useful cloning vectors. They are exclusively found in bacteria. Plasmids have also found their worth in being used as vectors for gene therapy in the near future.
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