1. Bacterial Transformation with (pGLO Plasmid)
Lab #9: Molecular Biology

2. Purpose of this Lab Learn how to insert a gene into bacteria
(Heat Shock)
Analyze how a gene can transform an organism and express that gene
Provide evidence that bacteria can take in foreign DNA in the form of a plasmid
Reinforce the following process:
DNA  RNA  Protein  Trait
Observe how genes are regulated

3. Applications of Genetic Transformation
Used in many areas of Biotechnology
Agriculture (pests, frost, & drought)
Bacteria (oil spills)
Gene therapy (sick cells into healthy cells)
Medicine (produce insulin & hormones)

4. Key Terms to Know DNA: Plasmid Bacteria: E. coli (strain: HB101K-12)
Growth media: LB Broth (Luria & Bertani)
Ampicillin: Antibiotic kills bacteria “amp”
Arabinose: Sugar source for energy & carbon
Heat shock Process that increases permeability of the cell membrane to DNA
GFP: Green Fluorescent Protein (w/UV)

5. The Genes of Interest Ampicillin resistance
Gene regulation proteins-activate the GFP gene when arabinose is present
GFP: Green Fluorescent Protein
-originally isolated from the jellyfish:
Aequorea victoria

6. Supplies for each Group of 4-5
(1) E. Coli-starter plate (incubator): has colonies present “LB”- S (Starter)
(4) Agar plates: 1-LB 2-LB/amp 1-LB/amp/ara
(in fridge)
Styrofoam holder (or ‘Jellyfish’ holder) to use w/ four colored Eppendorf vials (see next slide and/or white board). Label the vials with a sharpie
(5) Sterile Pipets and/or micropipets
(7) Yellow inoculation loops: 1 package (or nichrome wire…must sterilize EVERY TIME if using it)
Cup of crushed ice
(1) Sharpie marking pen
Masking tape
Water bath at 42 degrees C

7. Check your Materials-See List (NOTE- these colors may be DIFFERENT from what’s on hard copy of this PPt!!!
Take out what you’ll need
***(keep packages sealed)
Label your capped vials as follows:
Blue: “ - pGlo” (*CHANGE)
Green: “ + pGlo” (*CHANGE)
Pink: “TS”- Transformation Soln.(*CHANGE)
Yellow: “LB broth” (*CHANGE)

8. Using the Pipet Properly
Find the markings for each graduated volume on the pipet

9. Basic Process- READ before you start!
-Fill 100 ml beaker or cup with crushed ice. Set aside.
-USING GLOVES, obtain your ‘Starter’ colonies of E. coli (incubator). Get single colonies mm thick.
-Place a colony into each of the two tubes provided. MAKE SURE TO
LABEL: -pGlo and +pGlo (use new yellow loop EACH TIME)
-Add the pGlo plasmid to the +pGlo tube only (NEW yellow loop)
-Place tubes on ice (10 minutes- time this!!)
*bottom of tubes should be exposed to the ice
-Label the four plates as indicated in your guide
-Heat Shock the tubes in 42 C water bath using foam “floaters” for 50 SECONDS! (TIME THIS!) Have forceps ready to remove.
-Return to the ice (2 min- EXACTLY). Work quickly for best results!
-Add 250 L of LB broth to both tubes (sterile pipette each time)
-Add 100 L of tube contents to the appropriate plates
-Spread the samples (w/NEW yellow loop) & tape all four plates together with heat resistant tape (on cart). Put in incubator. CHECK MANUAL FOR CORRECT TEMP! Be sure group name is on dish

10. Adding Transformation Solution

11. Transferring Colonies, Labeling the Plates, & Using Heat Shock

12. The Process of Heat Shock
Helps to increase the bacterial uptake of foreign DNA
Membrane becomes more permeable to DNA
Time is essential:
-ice  water bath (42ºC) for 50 sec. ice
The number of transformants should increase

13. Adding the Bacteria to the labeled Plates

14. Expected Results PLATES OBSERVATIONS
+pGlo
LB/amp
Many colonies with white appearance
Transformation observed (resistance to amp)
NO fluorescence (No arabinose present)
LB/amp/ara
Many transformed white colonies
Fluoresce bright green under UV light
-pGlo
(CONTROL)
No Bacterial growth present on the plate
No transformation
LB only
(CONTROL
Bacteria present with whitish colonies
(regeneration of the starter plate)