AQA A Level Biology复习笔记8.4.5 Culture of Transformed Host Cells

In Vivo Method: Culture of Transformed Host Cells

 

  • 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

  • There are several steps involved in the process
  • A DNA fragment is isolated
    • The desired gene(s) is 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

     

  • 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

     

  • The vectors are transported into bacterial host cells
    • The cells containing the modified plasmids are described as transformed host cells

     

  • Bacteria multiply in number
    • Under the optimum conditions, the bacteria rapidly increase in numbers

     

  • 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 – genes that code for identifiable substances that can be tracked (eg. 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

     

  • The remaining bacteria are cultured
    • Every time a bacterium divides the desired gene is cloned

     

In-vivo-gene-cloning

The steps involved in the process of in vivo gene cloning using bacteria as the host cells

 

  • Recombinant DNA can be used to produce recombinant proteins (RP)
  • Bacteria have been genetically engineered for the production of human protein insulin to treat diabetes

2-Genetic-engineering-explained-12-Genetic-engineering-explained-22-Genetic-engineering-explained-32-Genetic-engineering-explained-4

An overview of the steps taken to genetically engineer an organism (in this case bacteria are being genetically engineered to produce human insulin)

 

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