1. pGLO Transformation LAB AP Investigation 8
ori
bla
GFP
araC
BIO-RAD lab book

2. Bio-Rad Video

3. Source of “glowing gene” for this experiment
Aequorea victoria:
Source of “glowing gene” for this experiment

4. Jellyfish Gene put into Other Critters

5. PLASMID Extrachromosomal DNA
Often carry genes for antibiotic resistance
Can be passed from one bacterium to another

6. Bacterial Transformation
The uptake of DNA
Bacterial Cell
Chromosomal
DNA
Plasmids

7. EXAMINE pGLO plasmid DNA
Use UV light to examine pGLO plasmid vial
UV light can be harmful to your eyes! Wear your goggles. Do not shine in eyes.
GFP = Green Fluorescent Protein
isolated from jellyfish
USED AS A GENETIC TOOL

8. LB (Luria and Bertani) – broth & agar
provides nutrients for bacterial growth
LB/amp
Luria agar + ampicillin (antibiotic)
LB/amp/ara
Luria agar + ampicillin + arabinose sugar

9. SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID)
OSMOTIC SHOCK =Transforming solution
CaCl2
HEAT SHOCK RAPID TEMPERATURE CHANGE is the key
50 SECONDS
2 MINUTES

10. pGLO plasmid ARABINOSE OPERON (INDUCIBLE)
Turns on when arabinose sugar is present
Allows bacteria to digest this sugar
pGLO
ori
bla
GFP
araC
Ori-
Plasmid
Replication
genes
GFP-Green Fluorescent Protein
- Glows green in fluorescent light
bla (beta-lactamase)
- On all time
- Makes protein that breaks down ampicillin
- Provides ampicillin resistance

11. ARABINOSE OPERON REGULATION
ara Operon
INDUCIBLE OPERON
PRESENCE OF ARABINOSE TURNS ON GENES
THAT MAKE ENZYMES
TO DIGEST ARABINOSE
araC B A D
Effector (Arabinose)
araC B A D
RNA Polymerase
araC B A D

12. pGLO Regulation ara Operon GFP GENE HAS BEEN ADDED TO ara OPERON
WHEN ARABINOSE
IS PRESENT,
OPERON IS
TURNED ON and GFP GENE IS EXPRESSED
TOO
Cells “glow” on media with arabinose
araC B A D
GFP Gene
Effector (Arabinose)
araC B A D
GFP Gene
RNA Polymerase
araC B A D
GFP Gene

13. Reasons for Each Transformation Step
Ca++ O
CH2 P
Base OH
Sugar
CaCl2 treatment
Positive charge of Ca+2 ions neutralizes:
negative charge of DNA phosphates
negative charge of membrane phospholipids

14. Reasons for Each Transformation Step
Incubation on ice slows fluidity cell membranes
Heat-shock increases permeability of cell membrane
Nutrient broth incubation allows beta lactamase expression

15. Selection for plasmid uptake
Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow on antibiotic (ampicillin) plate
only transformed bacteria grow
all bacteria grow a a a a a a a a a a a a a a a a a
LB plate
LB/amp plate
cloning

16. Transformation Results
LB PLATE Luria Broth +
- PGLO = NO Plasmid →
All cells grow since there is no antibiotic on the plate

17. Transformation Results
LB/AMP PLATE Luria Broth with antibiotic +
- PGLO = NO plasmid →
NO GROWTH Cells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

18. Transformation Results
LB/AMP PLATE Luria Broth with antibiotic +
+ PGLO = Plasmid added →
LAWN Cells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

19. Transformation Results
Cells with pGLO plasmid GROW & GLOW -can grow on media with antibiotic
GLOW on media with arabinose (turns on GFP gene)
LB/AMP/ARA PLATE Luria Broth + antibiotic| + arabinose +
+ PGLO = Plasmid added →

20. Labs due Monday 2-15 Lambda phage lab
Write out the procedure in your lab notebooks
For your data, DRAW a picture of the gel that your group had
If your data do not look like the picture I gave you, speculate why
Staple this to the questions/graph.
pGLO lab
Include the anticipated results of your lab – which plates would grow colonies and why
Answer the questions on pages 42-46