1. Lab 8 Warm-Up What are plasmids?
What gene(s) are on the plasmid being used in this lab?
How can we know if cells have been successfully transformed in this lab?

2. Warm-Up (Lab 8 Review)
What are the 3 genes found on the pGLO plasmid? What is the main function of each gene?
How can you tell if the E.coli bacteria successfully picked up the pGLO plasmid?
If the goal was to produce as much GFP as possible, what type of plate would you grow the E.coli on (what substances would be in the agar on the plate)?

3. Bacterial Transformation
AP Biology Investigation 8

4. Transformation Uptake of foreign DNA from surroundings
Plasmid: small ring of DNA that carries a few genes
Antibiotic resistance (eg. ampicillin)
AP Lab 8: transform pGLO plasmid into E.coli bacteria

5. pGLO Plasmid Beta Lactamase (bla): ampicillin resistance
Green Fluorescent Protein (GFP): jellyfish gene, glows under UV light
araC Regulator Protein: regulates GFP transcription; activated by presence of arabinose (sugar)

6. Transformation Lab
Luria Broth (LB) = liquid nutrients for bacterial growth
Transformation Solution (TS) = CaCl2, neutralize DNA backbone and phospholipids and allows DNA to enter cells
Heat Shock: increases permeability of cell membrane to DNA
+pGLO = solution contains pGLO plasmid
-pGLO = no pGLO plasmid

7. Predictions: Plate 1: LB/amp +pGLO Plate 2: LB/amp/ara +pGLO
Plate 4: LB –pGLO
Which plates will have bacterial growth?
Which plates will have bacteria that will GLOW?

8. Lab Instructions Read instructions CAREFULLY
Use sterile technique with petri dishes, pipettes and innoculating loops
Use a NEW pipette or loop for each specimen
Using pipettes: 100µl, 250 µl markings
Safety first: gloves, goggles, biohazard containers
Keep specimens ON ICE
Timing is Important!
Step #8: Heat Shock for 50 seconds
Step #10: Close tubes and flick to mix bacteria suspension before plating

9. Observations Draw what you see for each plate.
Plate 1: LB/amp +pGLO
Plate 2: LB/amp/ara +pGLO
Plate 3: LB/amp –pGLO
Plate 4: LB –pGLO
Count and record the number of colonies visible on each plate.
Note any other observations (color, etc.)

10. Analyzing Results
Do your results support your original predictions about the “+ pGLO” transformed E. coli cells versus “- pGLO” non-transformed cells?
Which of the traits that you originally observed for E. coli did not seem to become altered? Which traits seem now to be significantly different after performing the transformation procedure?
What evidence suggests that the changes were due to the transformation procedures you performed?

11. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?
Was your attempt at performing a genetic transformation successful? If so, how successful?
By calculating transformation efficiency, you can measure the success of your transformation quantitatively. Complete the transformation efficiency handout and attach in lab notebook.
What are some possible sources of error in the transformation procedure? If you had to repeat the procedure, what are ways to minimize potential sources of error?

12. Data Analysis: Calculating Transformation Efficiency
Total # colonies on plate/amt. DNA spread (μg)
Transformation efficiency (TE) is a way to describe how effective you were at getting a plasmid into bacteria.
The protocol we used generally has a TE between 8.0 x 102 and 7.0 x 103 transformants per microgram of DNA

13. Factors that affect transformation efficiency
Plasmid size (large vs. small)
Forms of DNA (supercoiled vs. relaxed DNA)
Growth of cells (competent cells)
Transformation protocol (heat shock time, temp., growth media, chemicals, contaminants)
Damage to DNA