viral gene cloning n.
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Viral Gene Cloning

Viral Gene Cloning

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Viral Gene Cloning

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  1. Viral Gene Cloning

  2. Phages can “hijack” bacterial replication machinery and clone a gene through self replication

  3. A phage can be used to insert a gene into a plasmid

  4. Verification of Gene Expression Techniques • GFP tagged protein • LacZ • Double Antibiotic

  5. GFP Expressed Ignore the Red Colonies Plasmid without GFP GFP Expressed GFP Marker • Gene of interest has GFP gene attached to it • Bacteria grown on antibiotic agar • Glowing colonies are expressing GFP and therefore your gene of interest

  6. LacZ • Plasmid has gene for Antibiotic resistance and lacZ. Gene of interest is inserted into the middle of lacZ to disrupt gene production. • LacZ encodes for the enzyme beta-galactosidase which breaks lactose into glucose and galactose. β-galactosidase cleaves the colorless substrate X-gal (included in agar) into galactose and a blue insoluble product of the cleavage • Colonies with gene of interest will grow white, colonies without gene will grow blue • Lactose can then be used to induce protein production

  7. Double Antibiotic • Plasmid vector contains genes for Antibiotic Resistance “A” and Antibiotic Resistance “B”. • Gene of interest is inserted into the middle of Antibiotic Resistance B to disrupt production • All bacteria with a plasmid will grow on a plate with Antibiotic A • A replica plate is made with Antibiotic B. Only empty plasmids without the gene of interest will grow • This plate is compared to the original plate and colonies with only resistance to Antibiotic A are selected

  8. In step 3 the different bacteria with these plasmid are cultured. Using a process called replica plating where a piece of velvet is put on top of the master plate bacteria, then transfered to make a copy onto the next. The cultures are next grown on a plate containing Ampicillin, this will kill the bacteria without the resistance to the gene, i.e bacteria with plasmid C (ring on insulin) and bacteria without our plasmid at all. What is left on the Amp plate will be bacteria with plasmids A and B (see diagram above this one). These are now transfered to a final plate containing Tetracycline. This will kill bacteria containing plasmid B because the Tetra. resistance gene has been disrupted by the insulin gene. Now all we have to do is compare the bacterial colonies to see where the insulin containing bacteria are. Follow this link for complete description of this technique