Features That are Required to Facilitate Cloning into a Vector
Origin of Replication (Ori)- Origin of replication (Ori) is a specific sequence of DNA bases which is responsible for initiating replication.
- A prokaryotic DNA has a single origin of replication while eukaryotic DNA may have more than one origin of replication.
- Some genes called "selectable markers" help in selecting those host cells which contain the vectors (transformants) and eliminating the non transformants.
- Transformation is a process through which a piece of DNA is introduced in a host bacterium.
- Generally, the genes encoding resistance to antibiotics such as tetracycline, ampicillin, kanamycin or chloramphenicol etc. are useful selectable markers for E. coli.
- The common E. coli cells are not resistant against any of these antibiotics.
- Plasmid PBR 322 has two resistance genes - ampicillin resistance ( amp ) and tetracyclin resistance ( tetr ) which are considered useful for selectable markers.
- Plasmid PBR 322 has a variety of unique recognition sites for restriction endonucleases.
- Two unique sites, Pst I and Pvu I are located within the amp gene and Bam HI, Sal I, etc. are within ter gene.
- Some other unique restriction sites are Eco RI, Cla I, Hind III, Pvu II, rop codes for the proteins involved in the replication of the plasmid.
- The presence of restriction sites within the markers tetR and amp permits an easy selection for cells transformed with the recombinant PBR322 .
- Insertion of the DNA fragment into the plasmid using enzyme Pst I or Pvu I places the DNA insert within the gene ampR this makes ampR nonfunctional.
- Bacterial cells containing such a recombinant PBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.
- Similarly when restriction enzyme Bam HI or Sal I is used, the DNA insert is placed within the gene tet r ' making it nonfunctional.
- Bacterial cells possessing such a recombinant PBR322 will, therefore, grow on ampicillin but not on tetracyclin.
- Due to inactivation of antibiotics, selection of recombinants becomes burdensome process because it requires simultaneous plating on two plates having different antibiotics.
- Thus, alternative selectable marker is developed to differentiate recombinants and non - recombi nants on the basis of their ability to produce colour in the presence of a chromogenic substance.
- Now a ' recombinant DNA is inserted in the coding sequence of an enzyme - galactosidase.
- This casues inactivation of the enzyme which is called insertional inactivation.
- If the plasmid in the bacterium does not have an insert, the presence of a chromogenic substrate gives blue coloured colonies.
- Presence of insert results into insertional inacti vation of the Beta galactosidase and, therefore, the colonies do not produce any colour, these colonies are marked as recombinant colonies.
- soil - inhabiting plant bacterium, Agrobacteriun tumefaciens, a pathogen (disease causing agent) of several dicot plants is able to transfer a piece of DNA known as ' T - DNA '.
- The T - DNA causes tumours.
- The tumours are called crown galls.
- Tumour formation is induced by Ti plasmid (Ti for tumour inducing).
- As gene transfer occurs without human effort, the bacterium is called natural genetic engineer of plants.
- Similarly retroviruses (cause leukosis or sarcoma types of cancer) in animals including humans are able to change normal cells into cancerous cells.
- The tumour including Ti plasmid of Agrobacterium tumefaciens have been modified into cloning vector which is not pathogenic to the plants, however,
- it is still able to use the procedure to deliver genes of our interest into various plants.
- Similarly retroviruses are used to carry desirable genes into animal cells.
- Thus once a gene or DNA fragment is joined to a suitable vector it is transferred into a bacterial plant or animal host where it undergoes multiplication.
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