Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
Copy Number in Vector
The copy number refers to the number of molecules of an individual plasmid/phage that are normally found in a single bacterial cell.
Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell.
Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.
If the foreign DNA (alien DNA) can be linked to this plasmid DNA or bacteriophage, the number of foreign DNA becomes equal to the copy number of the plasmid or bacteriophage.
In other words higher the copy number, more will be the gene expression, and hence more will be product obtained.
✼ Features of a Cloning Vector
Certain features are essential for the plasmid for the cloning to take place. These are as follows:
1. Origin of Replication (ori)
Sequence where replication starts.
If a DNA sequence is linked with the ‘ori’, it gets replicated.
‘Ori’ also regulates copy number of this linked DNA.
It helps in identifying the transformants from the non-transformants that can be eliminated.
It helps in selectively growing the transformants.
Transformation – The process through which a foreign DNA (plasmid/vector/rDNA) is introduced into a host bacterial cell.
Transformants – Bacterial cells that have successfully undergone the process of transformation and contains the foreign DNA.
pBR322
rop – it is the gene that expresses proteins that are essential for the replication of the plasmid.
Examples:
Antibiotic resistant gene
Ampicillin resistant gene (ampR)
Tetracycline resistant gene (tetR)
Kanamycin resistant gene
Chloramphenicol resistant gene
✼ Use of selectable Marker (Just for your Understanding!!)
It helps to distinguish a cell that has taken up a plasmid (transformant) from the many thousands that have not taken up the plasmid (non-transformants).
E. coli cells are normally sensitive to the antibiotics ampicillin and tetracycline.
However, cells that contain the plasmid pBR322 (one of the first cloning vectors to be developed) are resistant to these antibiotics.
This is because pBR322 carries genes, that makes the host cell (E. coli) resistant to ampicillin and tetracycline when expressed.
After transformation with pBR322, only those E. coli cells that have taken up a plasmid are ampRtetR and able to form colonies on an agar medium that contains ampicillin or tetracycline.
Non-transformants, which does not contain the pBR322, cannot express the antibiotic resistant genes, hence do not produce colonies on the agar medium that contains ampicillin or tetracycline.
Transformants and non-transformants are therefore easily distinguished.
Importance of Selection Marker
3. Cloning Sites
It refers to the segment of DNA in the plasmid where the alien (foreign) DNA can be inserted.
The vector/plasmid should ideally have one or very few recognition sites for the commonly used RE.
For the process of cloning, an RE is chosen that is generally part of the selection marker.
For example a foreign DNA can be ligated at the BamH I site of tetracycline resistance gene in the vector pBR322.
✼ Insertional Inactivation
When we grow bacterial cells on a selective medium (agar medium that contains ampicillin or tetracycline), we can differentiate between the transformants and non-transformants.
But we still have no idea if the transformants contains the recombinant plasmid DNA or the original plasmid DNA.
This technique is used to identify the recombinants from the non-recombinants.
Recombinants – Plasmid DNA with the inserted foreign/alien/target DNA.
The insertion of a foreign DNA fragment into the plasmid destroys the integrity of one of the genes (selectable marker gene) present on the molecule.
Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer displayed by the host cells.
Insertional inactivation of an antibiotic resistance gene
When a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322 is ligated, the recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA.
It can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium.
The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
The recombinants will grow in ampicillin containing medium but not on that containing tetracycline.
But non- recombinants will grow on the medium containing both the antibiotics.
Insertional inactivation using antibiotic resistance gene
Insertional inactivation without antibiotic resistance gene (β-galactosidase gene)
Here the recombinants and the non-recombinants are differentiated based on the basis of their ability to produce color in the presence of a chromogenic substrate.
In this method the foreign/target DNA is inserted within the coding sequence of an enzyme, β-galactosidase.
This inactivated the β-galactosidase gene expression.
When chromogenic substrate (X-gal) is added, bacterial colonies with functional β-galactosidase gives blue color, while bacterial colonies without the functional β-galactosidase gives no color.
Blue White Screening
✼ Nomenclature of pBR322 (Extra information)
“p” indicates that this is indeed a plasmid.
“BR” identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322).
“322” distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.).
✼ Vector for Cloning genes in Plants and Animals
For Plants
Agrobacterium tumifaciens : It delivers ‘T – DNA’ in the several dicot plants and transforms the normal cells into tumor and direct these tumor cells to produce the chemicals required by the pathogen.
The ‘Ti’ Plasmid of A tumifaciens has been modified into a cloning vector.
It is no more pathogenic to plants.
It can deliver the foreign gene into a large number of plants.
For Animal cells
Retroviruses : It can transform the normal animal cells into cancerous cells.
It now has been modified as follows:
It is no more pathogenic to animal cells.
It can deliver the foreign gene into animal cells.
❯ Introduction of Alien DNA into Host Cell
✼ Bacterial Transformation
Process of uptake of DNA by bacteria.
Competent Cells
As DNA is hydrophilic in nature, it cannot pass through the cell membranes.
All bacterial cells can not take up the desired DNA.
Only competent bacterial cells can take up the DNA.
These cells are prepared by treating them with a specific concentration of a divalent cation, such as calcium.[ 50 mM calcium chloride (CaCl2)]
It increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
Process of Transformation (Heat Shock Treatment)
Competent cells are incubated along with the rDNA (foreign/target DNA) in an ice-cold condition.
A heat shock is given to the cells by briefly placing them at 420C.
The cells are then again placed back on the ice.
This allows the competent cells to take up the foreign DNA.
Bacterial Transformation – Heat Shock Treatment
✼ Microinjection
Here the rDNA is directly injected into the nucleus of the host cell.
It makes use of a very fine pipette to inject DNA molecules.
This method is generally used for the animal cells.
✼ Biolistic / Gene gun
The host cells are bombarded with high velocity microprojectiles, usually particles of gold or tungsten coated with DNA.
This method is more suitable for the plant cells.
Microinjection and Gene gun Method of Gene Transfer
