8-4-5-culture-of-transformed-host-cells

Culture of transformed host cells

• The polymerase chain reaction (PCR) is a common molecular biology technique used in most applications of gene technology

  • It is described as the in vitro method of DNA amplification

• Gene cloning can also be carried out in vivo, using bacteria

  • Bacteria are the most common host cells for this method as they increase in numbers rapidly and are relatively easy to culture

In vivo gene cloning

1. A DNA fragment is isolated

   • The desired gene(s) are obtained by one of three methods

     • Extraction using restriction endonucleases

     • The conversion of mRNA to cDNA using reverse transcriptase

     • Artificial synthesis in a “gene machine”

   • Promoter and terminator regions are added to the fragments of DNA to ensure replication

2. The DNA fragments are inserted into vectors

   • Restriction endonucleases and ligase enzymes are used to insert the fragments of DNA into the vector

   • Plasmids are commonly used vectors

3. The vectors are transported into bacterial host cells

   • The cells containing the modified plasmids are described as transformed host cells

4. Bacteria multiply in number

   • Under the optimum conditions, the bacteria rapidly increase in numbers

5. Marker genes are used to identify the successfully transformed bacteria

   • Only a small fraction of the bacteria will have taken up the plasmid containing the desired gene

   • Those that have taken up the plasmid can be identified via markers

     • Markers are genes that code for identifiable substances that can be tracked (e.g. GFP – green fluorescent protein, which fluoresces under UV light, or GUS – a β-glucuronidase enzyme, which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent)

   • Those that have not taken up the desired gene are destroyed

6. The remaining bacteria are cultured

   • Every time a bacterium divides, the desired gene is cloned

Recombinant proteins

• Using the in vivo method above, recombinant DNA can be used to produce recombinant proteins (RP), such as insulin

  • Bacteria have been genetically engineered for the production of human protein insulin to treat diabetes