Bacterial transformation

Bacterial transformation

• 1.
  Bacterial Transformation RET Summer 2007
• 2.
  Overall Picture Bio-RadpGLO Transformation Insertion of GFP gene into HB101 E. coli
• 3.
  Transformation • The processof 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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  Selection • A selectivemedium 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.
  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.
  Factors that AffectYield 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.
  Transformation Applications
• 15.
  GFP Uses • Useas 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