1. Bacterial Transformation
RET Summer 2008

2. Overall Picture Bio-Rad pGLO Transformation Need: Good host
Self-replicating vector to carry our gene
Selection marker
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 & b-lactamase genes (contained in the pGLO plasmid)

4. Plasmids
Plasmids
small (1-200,000 kb)
circular
extrachromosomal DNA
“Relaxed” plasmids replicate independently 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
The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
DNA ligase joins the DNA fragment & vector DNA
Host cell is made competent so can plasmid can enter
Transformed cells are grown on selection media

6. Overall Transformation Process
The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
DNA ligase joins the DNA fragment & vector DNA
Host cell is made competent so can plasmid can enter
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
can leave blunt or sticky ends
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

8. Restriction Enzymes Insertion of a gene
Cut gene of interest with same restriction enzymes to make complementary “sticky ends”
DNA ligase:
joins the sticky ends of DNA fragments
Ideally, you would cut opposite ends of the gene with different restriction enzymes.

9. Overall Transformation Process
The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
DNA ligase joins the DNA fragment & vector DNA
Host cell is made competent so can plasmid can enter
Transformed cells are grown on selection media

10. Transformation of Bacteria
Generally occurs through heat shock and addition of a divalent cation
Calcium is thought to stabilize negatively charged phosphates
Heat shock briefly permeabilizes the membrane
Competent cells are those capable of taking up the plasmid
Cells most likely to become competent are in log growth phase

11. Overall Transformation Process
The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)
DNA ligase joins the DNA fragment & vector DNA
Host cell is made competent so can plasmid can enter
Transformed cells are grown on selection media

12. 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

13. 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)

14. 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

15. Transformation Applications

16. GFP Uses 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
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