1. BioRad pGLO: Transform bacteria with a Jellyfish gene to make them glow
Module based on a kit from
Bio-Rad Laboratories, Inc.
Thank you to :
Adapted by Dan Murray from a presentation by
Stan Hitomi
Monte Vista High School, Danville, CA.
Kirk Brown
Tracy High School, Tracy, CA.

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

3. Jellyfish Gene put into Other Critters

4. Outline Overview Bacteria and Plasmids Transformation The pGLO Plasmid
Experimental Procedures
Extension Activities

5. Overview

6. What is Bacterial Transformation?
Taking up of DNA from the environment by bacterial cells

7. Bacterial Transformation Lab
Bacterial Cells and plasmid DNA are mixed
Cells take up plasmid
Cell/DNA mix is plated on nutrient agar with antibiotic
Only cells which obtained plasmid DNA will grow… and glow

8. Bacteria and Plasmids

9. What is a plasmid? Small circular DNA molecule Replicates autonomously
Originally evolved in bacteria
May contain antibiotic resistance gene or be modified to contain other genes
bla is an ampicillin resistance gene
ori
bla

10. Bacterial Cells and DNA
Chromosomal
Bacterial cell
Chromosomal DNA
Plasmid DNA

11. Growth of Bacteria on Plates
Agarose in Petri dish = plate
Incubate at 37C
If few
cells grow
If many
cells grow
colonies
lawn

12. Transformation

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

14. Methods of transformation
Electroporation
Electrical shock makes cell membranes permeable to DNA
Calcium Chloride/Heat Shock
Chemically-competent cells uptake DNA after heat shock

15. The pGLO Plasmid

16. pGLO Plasmid bla gene GFP gene araC gene ori beta-lactamase enzyme
Ampicillin resistance
GFP gene
Green Fluorescent Protein
Aequorea victoria jellyfish
araC gene
On/off switch that reacts to arabinose
ori
Allows plasmid replication
pGLO
ori
bla
GFP
araC

17. pGLO Plasmid: Most Important Components
bla gene
Bacteria with this gene grow in the presence of ampicillin
GFP gene
Bacteria with this gene glow under near UV light
pGLO
GFP
bla

18. Experimental Procedures

19. Transformation Procedures
+CaCl2
+CaCl2

20. Transformation Procedures

21. Reasons for Each Transformation Step
Ca++ O
CH2 P
Base OH
Sugar
CaCl2 treatment
Positive charge of Ca2+ ions neutralizes:
negative charge of DNA phosphates
negative charge of membrane phospholipids

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

23. Purpose of each plate -pGLO/LB = Control
-pGLO/LB/amp = tests the effect of ampicillin
+pGLO/LB/amp = shows that ampicillin resistance has been acquired
+pGLO/LB/amp/ara = shows that both traits have been acquired

24. Transformation Results
Only cells getting pGLO plasmid grow and glow
CONTROL
All cells grow since there is no antibiotic on the plate
Without pGLO plasmid, nothing can grow
All cells grow since there is no antibiotic on the plate

26. Extension Activities

27. Extension Activity I: Transcriptional Regulation
Arabinose controls expression of GFP gene:
Transfer Bacteria
Glowing Bacteria from Transformation
Plate with
Arabinose
Plate without
Arabinose
Incubate 37C

28. Extension Activity I: Transcriptional Regulation
arabinose = no glow
+arabinose = glow
After overnight incubation
Plate with
Arabinose
Plate without
Arabinose

29. Transcriptional Regulation of GFP by Arabinose
araC
GFP Gene
araC repressor blocks transcription
Arabinose
araC
GFP Gene
Arabinose binds repressor, changing its conformation
RNA Polymerase
araC
GFP Gene
Altered repressor leaves DNA, RNA polymerase can perform transcription

30. Extension Activity II: Tweaking the Transformation Protocol
Test effect of various components of the transformation protocol:
plate ampicillin concentration
plate arabinose concentration
amount of plasmid DNA used in the experiment
amount of cells used in the experiment
length of time cells/DNA mix is kept at 42C during the experiment
Compare results with number of colonies
obtained during the normal protocol

31. Biotechnology
Explorer Program
Serious About Science Education