Plasmid

1) Self- replicating, double stranded, circular DNA molecule.

2) It’s maintained as an independent extrachromosomal entity in bacteria.

3) There are different types of plasmid that has been present in certain for survival and diversification:

• F plasmid à Involves in conjugation for transferring information to another bacteria. 

* Its total length may be 100 kb.
* It contains genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation.
* It has an insertion sequences that assist plasmid integration into the host cell chromosome.
(Small Picture of F plasmid.)

• R plasmid à resistance to antibiotic.

• Degradative plasmid à carry specific genes for the utilization of unusual metabolites.

• Cryptic plasmid à no functional coding genes. 

• Col plasmids à colicin production. Bacteriocin gene produces cloacins which kills bacteriophages. 

4) High copy number plasmids (relaxed plasmids) à 10-100 copies of plasmids per host cell.
5) Low copy number plasmids (Stringent plasmids) à 1-4 copies of plasmids per host cell.
6) Host range of plasmid is determined by its ori region. And there are two host range of plasmid i.e. Restricted where the plasmid is restricted to replicate in certain bacteria only (eg: Plasmid Col E1 replicates only in enteric bacteria like E.Coli or salmonella) and Broad host range where the plasmid can replicate any group of bacterium (RP4 plasmid can replicate in most of gram negative bacteria).

7) An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

Plasmid exists in three forms: covalently closed circles (CCC) DNA, open circles (OC) DNA, linear DNA.

Plasmids look like a coiled rubber band and . Topoisomerase, an enzyme that regulates the winding (over or under) of the DNA. It relaxes the supercoiled plasmid and brings CCC form. But if a Endonuclease (Enzyme cleaving the phosphodiester bond of DNA) attacks this uncoiled CCC, then a nick is formed in one of the strands of plasmid DNA. So therefore it forms a open circular DNA. The nick can be cured by another enzyme called DNA ligase which joins both the ends of the DNA strand. This enzyme is one of the important enzyme for joining the fragmented DNA with the plasmid. 

Now, DNA gyrase is type of Topoisomerase II which provide negative supercoiling of DNA by cutting the double strands and then joining them together by twisting the ends. 

In certain bacteria like streptomyces sp or Borrelia burgdorferi, plasmids are found in linear form. To protect the ends from nuclease digestion, there are two general mechanism involved:
a) terminal contains a hairpin loop structure (Borrelia).
b) terminal are protected by covalent attachment of protein (Streptomyces sp.)

Certain traits exhibited by the plasmid carrying genes

The R plasmid contains antibiotic resistance genes such 

The antibiotic resistance genes act as marker for identifying the plasmids containing the gene of interest. For eg: plasmid containing Ampicillin resistant marker will let the host bacteria to grow in a medium containing ampicillin. 

Plasmid protects the  bacterium for hazardous condition and enable them to survive even in limited resources. They also pass some part or whole gene to another host genome or plasmid through conjugation.  

Plasmid in gene cloning

What makes the plasmid a suitable vector for gene cloning?

* Plasmids are easy to isolate, purify and can be reintroduced into a bacterium by transformation.

* They bear antibiotic resistant genes which can be used for selecting the bacterial host during screening process.

* Plasmids with a foreign DNA is said to be chimera.

* They contain origin of replication (ori) region that can allow the plasmid to replicate individually from the bacterial genome.

* plasmids with high copy number, low molecular weight and conjugation property can be produced in more numbers and thus increasing the volume of sample. 

pBR322 – First gene cloning vector

1. It contains 4,361 bp and carries two antibiotic resistance genes: Ampr (Ampicillin) and Tetr (Tetracycline).

2. Plasmid make up: Within Tet^r (BamH1, HindIII, and SalI RE recognition site), Within Amp^r (PstI site), EcoRI not within any coding DNA, and origin of replication that functions only in E.Coli.

How does this pBR322 plasmid work as cloning vector? 

How does the Alkaline phosphatase treated linear plasmid ligate together even with lose of phosphate group at 5' end? 

A phosphate group at the 5' end is important for the T4 ligase to join the plasmid and the target DNA. But due to the loss of 5' phosphate group, two nicks are formed at the joining site. Therefore the plasmid with target DNA should become unstable and linear. 

Somehow this doesn't happen, because two phosphate group present in the target DNA hold the plasmid together and once they are transformed into the host cell, the two nicks are ligated by the host DNA ligase enzyme. 

Other plasmids

Ti plasmids