1. Bacterial Transformation RET Summer 2007

2. Overall Picture Bio-Rad pGLO Transformation Insertion of GFP gene into HB101 E. coli

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. 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 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. Selection Media LB plates: LB + amp: LB + amp + ara: Control (-pGLO) Should contain only cells with the amp- resistant pGLO plasmid; colonies appear white (-pGLO, + pGLO) Should contain only cells with the amp-resistant pGLO plasmid; colonies floresce green (+pGLO)

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. Transformation Applications

15. GFP Uses Use as a reporter molecule to follow changes in gene expression over time Nondestructive, nontoxic Coding sequence can be cloned into a variety of vectors GFP keeps its fluorescence in cells from different species Can be tracked in living cells over to time to study development Can be directed to specific subcellular compartments Can combine GFP coding region with the regulatory region for another gene and observe changes in gene expression Can be used to make a fusion protein to study localization, turnover & intracellular associations of native protein GFP gene is switched on when cells are grown in the presence of arabinose