Introduction of Vector Into Hosts

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Chapter: Pharmaceutical Microbiology : Recombinant DNA Technology

For the expression and maintenance of recombinant genes the recombinant vectors harbouring them need to be introduced into suitable hosts. The four main methods used to achieve this are transformation, electroporation, conjugation and transduction.


INTRODUCTION OF VECTOR INTO HOSTS

 

For the expression and maintenance of recombinant genes the recombinant vectors harbouring them need to be introduced into suitable hosts. The four main methods used to achieve this are transformation, electroporation, conjugation and transduction.

 

• Transformation is the direct incorporation of DNA into host cells. Bacteria such as E. coli can uptake recombinant plasmid DNA when treated with ice-cold CaCl2 until they reach a ‘competent’ state in which they are ready to take up DNA. These cells are then mixed with the recombinant plasmid and exposed briefly to a heat shock of 42°C which causes them to take up the DNA.

 

• Electroporation is, however, the most efficient way of introducing DNA not only in bacteria but also in eukaryotic cells. This technique is based on the induction of free DNA uptake by the cells after subjecting them to a strong electric field.

 

• In some cases conjugation can be used as a natural transmission of plasmid DNA from a donor cell to a recipient cell by direct contact through cell–cell junctions. Only plasmid cloning vectors containing conjugative elements can be transferred by conjugation. This procedure requires direct contact between the donor and the recipient cell. Conjugation is not as frequently used as electroporation as most plasmid vectors used for the cloning of recombinant DNA lack conjugative functions, preventing these plasmids from being passed to other cells inadvertently.

 

• Finally, in transduction (prokaryotes) and transfection (eukaryotes) the transfer of recombinant non-viral DNA to a cell is achieved by a virus. This is the method of choice for the introduction of recombinant λ bacteriophages, cosmids and fosmids into E. coli cells.

 

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