1. Mrs. Stewart Medical Interventions Central Magnet School
Transformation Lab
Mrs. Stewart
Medical Interventions
Central Magnet School

2. Purpose
We will demonstrate the ability to transform bacterial cells with free-floating DNA using a gene that codes for Green Fluorescent Protein (GFP).

3. Transformation
The genetic modification of a bacterium by the incorporation of free-floating DNA from the surrounding environment

4. GFP – Green Fluorescent Protein
Gene found in bioluminescent jellyfish Aequorea victoria
Gene causes jellyfish to fluoresce and glow in the dark
If transformation is successful, bacteria will produce this protein and fluoresce under UV light

5. pGlo Plasmid DNA Ori = origin of replication
araC = arabinose detection gene that regulates the production of GFP
Bla = beta – lactamase enzyme gene (antibiotic resistance to beta-lactam abx)
GFP = green fluorescent protein gene inserted into plasmid using restriction enzymes

6. Introduction to pGlo Gene Regulation
Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the simple sugar arabinose is both a source of energy and a source of carbon for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off again. Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. When bacteria that have been transformed with pGLO plasmid DNA are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow brilliant green when exposed to UV light.

7. Day One – Materials Student Stations Common Materials LB agar plate
Alcohol burner
Permanent marker
Inoculation loop
Common Materials
E. coli
Transformation solution

8. Day One – Streak a Starter Plate
The purpose of this starter plate is to have SINGLE COLONIES
Streak for colonies

9. Day Two – Materials Student Stations Group E. coli starter plate
2 – 1.5 ml micro test tubes + tube rack
4 - agar plates – LB, 2 LB/amp, LB/amp/ara
Transformation solution
LB broth
Inoculation loops
Micropipet (.5-10 µl) + tips
3 - Sterile disposable pipets
Permanent marker

10. Step 1: Labeling Label the 2 micro test tubes “+ pGlo” on one tube
Add group name to both
Place tubes in tube rack

11. Step 2
Transfer 250 µl of transformation solution (CaCl2)into each tube*
Place tubes on ice to maintain sterility
*Purpose: Both DNA and a bacterial cell wall have a negative charge. CaCl2 will help neutralize those opposing charges to increase change of DNA uptake

12. Step 3
Use a sterile loop to pick up 3 or 4 LARGE colonies with a uniformly circular shape and smooth edges.
Do not use a swab of bacteria from the dense growth – we need actively growing bacteria for optimum transformation
Transfer loop w/ bacteria into +pGlo tube and spin until colony is dispersed.
Repeat for –pGlo tube

13. Step 4 Examine the pGlo DNA solution with the UV light
Record observations
Transfer 10 µl pGlo DNA solution into +pGlo tube
DO NOT add pGlo plasmid DNA to –pGlo tube
Incubate on ice for 10 minutes
10 minutes

14. Step 5 Label the 4 agar plates as follows:
Add group name to all 4 of them
Label 2 plates +pGlo - LB/amp and LB/amp/ara
Label 2 plates –pGlo – LB/amp and LB

15. Step 6 - HEAT SHOCK
Transfer both tubes from ice to heat block (42oC) for exactly 50 seconds
Immediately transfer back to ice after the 50 seconds has ended
Incubate on ice for 2 minutes

16. Step 7 Remove tubes from ice
Add 250 µl of LB nutrient broth to each tube using a sterile pipet
Incubate at room temp for 10 minutes

17. Step 8
Gently flick/tap tubes with your finger to mix and resuspend bacteria
Pipet 100 µl of transformation of each tube onto the corresponding plates

18. Step 9 Use a new sterile loop for each plate
Spread the suspensions evenly around the plate surface
DO NOT puncture the agar by pressing too deep

19. Step 10 Stack plates and tape together Incubate
3rd period – countertop
2nd period – incubator
Store them upside down!