Sunday, 7 March 2021

Plasmid - Defination, Structure, Properties, replication, Copy number, Types, Functions and applications

Plasmid is a short, naturally occurring extra chromosomal, usually circular, double stranded DNA molecule that replicate, autonomously and lead an independent existence in Bacterial cell.

The term Plasmid was first given by Joshua Lederberg in 1952.

Properties of Plasmid :

• Plasmid is naturally found in cytoplasm, seperately from the main bacterial chromosome and are much smaller incomparision.

• Plasmids are mostly circular negatively supercoiled, double stranded DNA molecule. But linear Plasmids are also reported in genera streptomycetes and Borrelia. Also RNA plasmids are rare but reported in few plants, fungi and animal.

• Plasmids are normally in size range of 1kb to 250 kb which is smaller much smaller than Bacterial chromosome.
• Plasmids are considered as replicon. They replicate independently and code for their own transfer (i.e. Ori site present).
  Smaller plasmid use host cell DNA replication machinary.
  Larger plasmid carry gene for special enzymes specific for plasmid replication.

• Some are interogative plasmid called episomes. Also able to replicate by inserting them into bacterial chromosome and may be stably maintained in this form through numerous cell division, but show independent existence at some stage.

Copy number : Number of plasmids in an individual bacterial cell may very (1-100 or more) denoted by copy number.

• Plasmids are not essential for viability of bacterial cell. but genes carried by plasmids usually encodes traits beneficial for bacteria. e.g. antibiotic resistance, heavy metal resistance, Virulence factors, N2 fixation.

• Plasmid provide a mechanism of horizontal gene transfer via conjugation, transduction and transformation
Examples of plasmids : Puc 8 (E.cli), R-1, R-6, Col E1 (E.coli), Tol (Pseudomonas putida).

General Structure of Plasmid :

  Structurel elements of bacterial plasmids may vary according to their size and function. Plasmid have following elements -

Structure of Plasmid

1. Origin of replication : DNA sequence which encode initiation of plasmid replication by recruiting bacterial transcription machinary for replication enzymes & proteins.

2. Antibiotic resistance gene : these genes provide a survival advantage to the bacterial host thet allows for selection of plasmid containing bacteria.

3. Multiple cloning site : Short segment containing a several restriction enzyme sites enabling easy insertion of foreign DNA.

4. Promoter region : It drives the transcription of the foreign insert.

5. Selectable marker : It is used to select for cells that has successfully taken up plasmid for the purpose of expression of inserted DNA.

6. Primer binding site : It is used as an initiation point for PCR amplification or sequencing of the plasmid.

Types of Plasmid (based on function) -

A. Fertility or F-plasmid :

- It carry only transfer (tra) genes.
- It have no characteristic beyond the ability to promote conjugal transfer of plasmids. F plasmid is also called episomes.
- Responsible for conjugation hence called conjugation factors.
F plasmid integrates with chromosomes to form Hfr+
- Both ori-V and ori-T present
E.g.  F plasmid of E.coli.

B. Resistance or R-plasmid :

This plasmids carry genes thet provide resistance to one or more antibiotics and poisons. e.g.  Chloremphinicol, ampicillin, tetracycline, mercury etc.
Example of this type plasmid is Rp4 in Pseudomonas.

C. Col plasmid :

This plasmid carry genes that codes for colicins proteins that kill other bacteria.
E.g. Col E1 in E.coli.

D. Metabolic/Degradative Plasmid :

This types of plasmid carry genes that allow the host bacterium to metabolize unusual substances like Toluene and salicylic acid.
E.g. Tol plasmid of Pseudomonas putida.

E. Virulence Plasmid :

This plasmid carry genes that turn host bacterium into pathogen
E.g. Ti plasmid of Agrobacterium tumifaciens, which induce crown gall disease on dicot plants.

F. Suicide plasmid :

It transfer to another bacterial cell and do not replicate further.

Types of plasmid based on their ability to transfer to another bacteria -

A. Conjugative plasmid - This plasmid contain tra gene for conjugation and transfer of plasmid to another bacterium (tra+, mob+).
E.g. F+ plasmid and many R plasmid and some Col plasmid.

B. Non-conjugative plasmid -
This plasmid incapable of initiating conjugation, hence can only be transferred with the help of conjugative plasmid (tra-, mob-) under some circumstances.
E.g. many R plasmid, most Col plasmid.

C. Mobilisable plasmid :
This is an intermediate class of plasmid carry only a subset of genes (mob+) required for transfer.
They parasitize another plasmid, transferring at high frequency in presence of conjugative plasmid
(tra+, mob+).

Plasmid replication :

Plasmid are replicate autonomously and it also contain origin of replication site. There are two models are proposed for plasmid replication -
1) Theta θ model :
2) Rolling circle model :

1). Theta θ model :

- Mostly plasmids in gram negative bacteria replicate in this manners.

Theta model replication of plasmid
- Similar to replication of bacterial chromosome.
- This plasmids have an origin of replication ori-V called origin of vegetative replication where DNA opens up.
- Bidirectional replication around the DNA circle occurs giving a theta(θ) intermediate.

2). Rolling circle model :

- Followed by some plasmids such as F plasmids of E.coli during conjugation. Such plasmid has two separate ori sites :
- Ori-V  starts vegetative replication.
- Ori-T  transfer starts from ori T one strand and using other still circular as templete.

Host range :

There is mainly two types of host range
1. Narrow host range -
Most of the plasmids can exist in a limited number of host bacterial species.
2. Broad host range - Can be transferred and maintained in large number of bacterial species
E.g. Inc P of E.coli
These are generally used for cloning as vectors.

Copy Number :

Copy number refers to the number of plasmid molecules of an individual.
Plasmid that are normally found in a single bacterial cell.
Plasmids are Classifying into two types based on copy no.
1). Stringent Plasmid :
- It is a large plasmid molecule.
- have low copy no. It present 1 to 2 per cell.
2). Relaxed plasmid :
- This plasmids are very small.
- have high copy no. 50 or more per cell.

Regulation of copy number :

Copy number is regulated by negative regulatory mechanisms and adjusting rate of plasmid replication.

1, by antisense RNA counter transcribes RNA,- plasmid replication is controlled by regulating the amount of available primer for replication.

2, Regulation by binding of replication proteins to introns (18-22 bp repeated units). Regulating amount of replication machinary available.

3, Regulation by ct RNA and protein, counter transcribed RNA limits the function of essential replication protein.

Plasmid incompatibility :

Incompatibility is defined as inability of two closely related plasmids to co-exist stably in the same host cell.
Several different kinds of plasmids may be found in a single cell, including more than one different conjugative plasmid.
To be able to coexist in the same cell, different plasmids must be compatible.
Different types of plasmids can therefore be assigned to different incompatibility groups, on the basis of whether or not they can coexist.

Incompatibility group : two plasmids that can not coexist in the same bacterial cell belong to a incompatibility group.

Why plasmids are incompatible ?

  Because incompatible plasmids share same replication control machanism (RpCM).
So same (RpCM) does not recognise 2 plasmid as different.
So it select either one is randomly selected and eventually progeny contains either one which is selected by replication control machanism.

Partitioning :

It is active process that ensure that after cell division each daughter cell gets at least one copy of plasmid.
- Relaxed plasmid each daughter cell after division may get a copy by random diffusion or segregation.
- Stringent Plasmid most likely one daughter cell not received plasmid during segregation.

Par ABS system :- it is a broadly conserved molecular mechanisms for plasmid partitioning and chromosome segregation in bacteria.

Par ABS has 3 elements -
Par A - ATPas
Par B - DNA binding protein
Both are found on same operon.
Par S - cis acting sequence, located within or adjacent to this operon.

Confirmation of Plasmids :

- Nicked open circular DNA
- Relaxed circular DNA
- Linear DNA
- Supercoiled or Covalently closed circular DNA
- Super coiled denatured DNA

Functions/Uses of plasmid (in genetic engineering) :

1. Cloning : to multiply & express particular genes
2. Protein production e.g. insulin
3. Gene therapy : Plasmid vactor used for insertion of therapeutic gene at Target site in human genome.
4. Genetic disease models : genetically engineered the, embryonic stem cells of rat.
5. Use for Plasmid DNA vaccines.

Advantages of using plasmid in genetic engineering :

1. Easy to work with and modify due to convenient size.
2. It contain independent origin of replication site.
3. Easy amplification due to their multiple Copy number.
4. Presence of selectable markers makes detection and selection of clones easier.
5. Stable for longer time as glycerol stocks.
6. Function in many species e.g. plants, mice and even cultured human cells.

Plasmid in eukaryotes :

- circular plasmid which is 6.3 kb circular found in most Saccharomyces cerevisiae strain.
- 50-100 copies per haploid genome of yeast.
- This is the mostly studied eukaryotic plasmid.




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