Plasmids are extrachromosomal DNA molecules. They are small, circular and have an ability to replicate autonomously. Replication of plasmid is not under control of chromosomal DNA. They are mostly found in bacteria. Some of the eukaryotes like yeast and plants also contain plasmids.
Their ability to replicate independently makes plasmid a cloning vector in the recombinant DNA technology for transferring and manipulating genes.
Many antibiotic resistance genes in bacteria are present in plasmids.
The size of plasmid varies from a few base pairs to thousands of bp.
Plasmids also get transferred from one bacterial cell to another by the process of conjugation.
Plasmids carrying a specific gene are introduced into bacteria cells, which multiply rapidly and the required DNA fragment is produced in larger quantities.
Plasmids are used to prepare a recombinant DNA with the desired gene to transfer genes from one organism to another. This is known as genetic engineering.
Joshua Lederberg coined the term plasmid.
- Plasmids are extrachromosomal and not essential. They are useful but not necessarily present in every organism of the species
- Plasmids are not a part of the genome and the same plasmid can exist in different species and gets transferred from one another
- Plasmids have their own origin of replication (ORI) and they replicate along with the cell so that each daughter cell possesses a copy of the plasmid also
- Apart from the origin of replication, often it contains genes for antibiotic resistance, for the production of toxins and other useful genes, that may be required for the survival of cells
Plasmids and bacteriophages are frequently used as a cloning vector in the DNA recombinant technology.
- The ease with which plasmids can be modified and replicated makes it a great tool in genetic engineering and biotechnology
- For genetic engineering purpose, plasmids are artificially prepared in the lab
- The lab-grown plasmids, which are used as a vector contain an origin of replication, cloning site and selection marker
|Origin of Replication
|DNA sequence where initiation of replication starts|
|Selectable Marker||For selecting bacteria containing desired plasmid, e.g. antibiotic resistance genes and other specific genes|
|Multiple Cloning Sites (MCS)||Recognition sites to insert foreign DNA fragment by using restriction enzymes, a few or single recognition site is preferred to avoid getting several fragments|
|Promoter Region||Promotes transcription of the target gene to get the desired protein|
|Primer Binding site||The sequence of DNA used as a start point for PCR amplification and sequence verification|
- DNA is cut at the specific points by using restriction enzymes (molecular scissors), which make sticky ends of the DNA
Restriction enzymes were discovered by Herbert Boyer
Herbert Boyer and Stanley Norman Cohen together discovered recombinant DNA technology by recombining DNA segments as desired and inserting into the bacteria cell to get the desired protein
- The desired genes are then inserted by using DNA Ligase
- The recombinant DNA molecule is then introduced to the host bacteria cell by the process of transformation
- The recombinant plasmid then multiplies using host DNA polymerase
- The first plasmid used as a cloning vector was pSC101 of Salmonella typhimurium. They showed that a gene from a frog can be expressed in the bacterial cell
- E.coli. plasmid is frequently used as a cloning vector
The main characteristics of a plasmid cloning vector of E.coli, pBR322 are:
- Restriction sites: Hind III, BamH I, Sal I, Pvu II, Pst I, Pvu I, EcoR I, Cla I
- Selectable marker: antibiotic resistance genes for ampicillin (ampR) and tetracycline (tetR)
- ORI: the origin of replication
- ROP: codes for proteins, which are involved in the process of replication of plasmid
One of the antibiotic resistance genes act as a restriction site and to ligate foreign DNA and another is used for the selection of transformants. The gene, where the foreign DNA is inserted becomes inactive.
Alternative selectable marker: Mostly these have an ability to produce some colour after reacting with a chromogenic substance. The alternative markers are used for the ease of differentiating recombinants from non-recombinants, e.g. gene coding for β-galactosidase.
When a foreign gene is inserted between the gene coding for β-galactosidase, the recombinant cell does not produce the enzyme β-galactosidase due to inactivation of the gene. In the presence of a chromogenic substrate, non-recombinants produce blue colour colonies and recombinants produce colourless colonies.
The Tumour inducing or Ti plasmid is present in the bacterium Agrobacterium tumifaciens.
It is widely used now as a cloning vector to deliver desirable genes to the host plant to get transgenic plants. The main characteristics of Ti plasmid are:
- Size of the plasmid is ~ 250kbp
- There are different kinds of Ti plasmids based on the different genes they possess, which code for different opines, e.g. leucinopine, nopaline, octopline, etc.
- It is a pathogenic species to many dicot plants. It causes crown gall disease in plants.
- It contains one or more T-DNA region
- Agrobacterium tumifaciens has an ability to transform the normal cells to tumour cells by inserting a DNA piece known as T DNA and it starts producing chemicals required by the bacterium
- After inserting the desired gene into Ti plasmid, it loses its pathogenic ability but is still able to insert the desired gene into the plant cell
- It contains vir or virulence genes, which transfer T-DNA region to plant cells and gets integrated into the plant genome
- Ti plasmid can be modified as per the requirement to insert the desired genes
- Agrobacterium tumifaciens is called “nature’s genetic engineer”