From Genotype to Phenotype

• Focus of Week 2: GFP (Green Fluorescent Protein)

Transformation of Competent Cells

• Transformation Process:
  • Introduction of plasmids into competent E. coli cells.
  • Competent cells are prepared to uptake foreign DNA.
• Goals:
  • Understand the significance of transformation.
  • Discuss why two different plasmids were used.

Plasmids Used

• pGLO Plasmid
  • Components:
  • ori: Origin of replication
  • araC: Gene for arabinose regulatory protein (bifunctional)
  • PBAD: Promoter region for arabinose-induced expression
  • GFP: Gene encoding Green Fluorescent Protein
  • bla: ẞ-lactamase gene for ampicillin resistance

Experimental Predictions

• Expected Growth on Plates:
  • Identify which plate will have the most bacterial growth.
  • Determine how to confirm transformation success.
  • Define control plates and their purposes:
  • Media:
    • LB: (no antibiotic)
    • LB/Amp: (with ampicillin)
    • LB/Amp + plasmid: (with plasmid, either +pUC19 or +pGLO)
  • Expectations:
  • Growth on LB and LB/Amp plates but not LB/Amp with dH2O.

Results Interpretation

• Discussion of Results from Previous Experiment:
  • Compare results with predictions.
  • Explain colony formations:
  • High number of colonies on the dH2O LB plate due to no selective pressure.
  • Fewer colonies on +pUC19 and +pGLO plates due to antibiotic selection.
  • Analyze results on LB-Amp plate - are there colonies, and should there have been?
  • Identify new phenotypes resulting from genetic changes.
  • Define what constitutes a bacterial colony and its formation process.

Restriction Enzymes

• Function:
  • Restriction enzymes cut DNA at specific sequences.
  • Example: EcoRI cuts at the sites:
  • Sequence:
    • 5’-GAATTC-3’
    • 3’-CTTAAG-5’
  • Used to cut plasmid DNA at engineered "cut sites."

Restriction Digestion of pGLO Plasmid

• Materials:
  • pGLO plasmid, restriction enzyme (EcoRI or EcoRI & NheI), buffer (Mg2+), and distilled water
  • Incubation:
  • For 1 hour at 37°C.

Visualization of DNA

• Technique:
  • Agarose Gel Electrophoresis
• Process Overview:
  • DNA is subjected to an electric field:
  • Cathode (Negative) and Anode (Positive)
  • DNA fragments migrate through the gel based on size.

Types of Plasmids After Purification

• Forms of Plasmid:
  • Supercoiled, relaxed circular, nicked circular
• Electrophoresis Characteristics:
  • Different forms affect migration speed in gel.

Size Marker and Gel Patterns

• size markers:

  • Indicate fragment sizes visualized during agarose gel electrophoresis
  • Sizes: Examples include 500 bp and 3 kb fragments.

• Sample Results:

  • Comparison of no enzyme, EcoRI, and EcoRI + NheI to understand restriction effects on DNA size and shape.