F215 OCR Genetic Engineering

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  • Genetic Engineering
    • eg Insulin & Golden Rice
    • 1. The Required Gene Is Obtained
      • - Use mRNA (Avoids finding gene amongst entire human genome
        • - From B cells in the IOL. Transcribe and Translate the Human Insulin Gene
          • - Isolate mRNA & add reverse Transcriptase= produces cDNA which is single stranded
            • - Add DNA Polymerase & free nucleotides to join SP Backbone add DNA Ligase (phosphodiester bond) to form DNA
      • Use Restriction enzymes & find with gene probe
    • 2. A copy of the gene is placed into a Vector
      • What is a Vector? A vehicle that carries the gene of interest into target cell
      • Placed in Plasmid, small circular loop of DNA with antibiotic resistance that can self replicate
        • Cut open using Restriction Enzymes, create sticky end that are complementary to insulin gene
          • Add artificial sticky ends onto insulin gene
            • H Bonds form between complementary bases= 2 A&T, 3 G&C
              • RecombinantPlasmid formed = Joining of DNA from 2 different species
    • 3. The vector carries gene into Recipient Cell = E.Coli
      • Bacteria grow on agar plate don't know which ones have taken it up so we use Marker Genes
        • Genes that allow you to identify & select bacteria that have taken up the gene of interest eg insulin
          • Plasmids have two different genes Ampicillin & Tetracycline
            • Insulin in the vector breaks the tetracycline gene so no longer resistant but still resistant to ampicillin
        • Process = Replica Plating. Ampicillin agar all bacteria grow then transfered to Tertracycline agar only plasmid without insulin will grow
          • By keeping track of the colonies the desirable colonies can be identified
    • 4. Transformed bacteria contaning insulin gene are grown on a large scale in a fermenter
      • Bacteria is transcribed and translated and insulin polypeptide is produced then extracted and purified
      • Protein Synthesis
        • Bacteria is transcribed and translated and insulin polypeptide is produced then extracted and purified




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