F215 OCR Genetic Engineering
- Created by: ellielewis98
- Created on: 21-05-16 21:22
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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
- - Isolate mRNA & add reverse Transcriptase= produces cDNA which is single stranded
- - From B cells in the IOL. Transcribe and Translate the Human Insulin Gene
- Use Restriction enzymes & find with gene probe
- - Use mRNA (Avoids finding gene amongst entire human genome
- 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
- H Bonds form between complementary bases= 2 A&T, 3 G&C
- Add artificial sticky ends onto insulin gene
- Cut open using Restriction Enzymes, create sticky end that are complementary to insulin gene
- 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
- Plasmids have two different genes Ampicillin & Tetracycline
- 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
- Genes that allow you to identify & select bacteria that have taken up the gene of interest eg insulin
- Bacteria grow on agar plate don't know which ones have taken it up so we use Marker Genes
- 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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