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Bacterial transformation

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Bacterial transformation

  1. 1. Bacterial Transformation RET Summer 2007
  2. 2. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli
  3. 3. Transformation • The process of transferring foreign DNA fragments into a recipient (host) cell for growth and replication • Our host cells: HB101 E. coli • Our foreign DNA: GFP & -lactamase genes (contained in the pGLO plasmid)
  4. 4. Plasmids • Plasmids – small (1-1000 kb) – circular – extrachromosomal DNA • Growth is independent of the host’s cell cycle; amplification of gene product • A type of cloning vector used to carry a gene not found in the bacterial host’s chromosome
  5. 5. Overall Transformation Process 1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme) 2. DNA ligase joins the DNA fragment & vector DNA 3. Host cell is made competent so can plasmid can enter 4. Transformed cells are grown on selection media
  6. 6. Overall Transformation Process 1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme) 2. DNA ligase joins the DNA fragment & vector DNA 3. Host cell is made competent so can plasmid can enter 4. Transformed cells are grown on selection media
  7. 7. Restriction Enzymes • Endonucleases: – in nature, they protect bacteria from intruding DNA – cut up (restrict) the viral DNA – cut only at very specific nucleotide sequences • Restriction site: recognition sequence for a particular restriction enzyme • Restriction fragments: segments of DNA cut by restriction enzymes in a reproducible way • DNA ligase: joins the sticky ends of DNA fragments
  8. 8. Overall Transformation Process 1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme) 2. DNA ligase joins the DNA fragment & vector DNA 3. Host cell is made competent so can plasmid can enter 4. Transformed cells are grown on selection media
  9. 9. Transformation of Bacteria • Generally occurs through heat shock and addition of a divalent cation to permeabilize the membrane • Competent cells are those capable of taking up the plasmid
  10. 10. Overall Transformation Process 1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme) 2. DNA ligase joins the DNA fragment & vector DNA 3. Host cell is made competent so can plasmid can enter 4. Transformed cells are grown on selection media
  11. 11. Selection • A selective medium is used to determine which bacterial cells contain the antibiotic resistant plasmid insert and which do not • For example, a bacterium containing a plasmid with resistance to a particular antibiotic (ampicillin) will grow on medium that contains that antibiotic • In addition, our plasmid contains a regulatory element that activates the GFP gene only in the presence of arabinose
  12. 12. Selection Media LB plates: Control (-pGLO) LB + amp: Should contain only cells with the amp- resistant pGLO plasmid; colonies appear white (-pGLO, + pGLO) LB + amp + ara: Should contain only cells with the amp-resistant pGLO plasmid; colonies floresce green (+pGLO)
  13. 13. Factors that Affect Yield and Quality of Plasmid DNA • Plasmid copy number • Host strain used, carbohydrate production • Culture medium, selection, and culture time – Want to harvest during log growth phase
  14. 14. Transformation Applications
  15. 15. GFP Uses • Use as a reporter molecule to • Can be directed to specific follow changes in gene subcellular compartments expression over time • Can combine GFP coding region with the regulatory • Nondestructive, nontoxic region for another gene and • Coding sequence can be observe changes in gene cloned into a variety of expression vectors • Can be used to make a fusion • GFP keeps its fluorescence in protein to study localization, cells from different species turnover & intracellular associations of native protein • Can be tracked in living cells • GFP gene is switched on over to time to study when cells are grown in the development presence of arabinose

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