1. Pre-Lab: pGLO Bacterial Transformation
AP Biology
Ms. Day

2. Bacterial Transformation
Step 1 DNA Isolation
Isolation of the “Gene of Interest” (foreign DNA)
Step 2 Recombinant DNA
Insertion of foreign DNA into bacterial plasmid using restriction enzymes and DNA ligase
Step 3 Transformation
Insertion of recombinant DNA into bacteria by making bacteria competent (weaken)
Use CaCl2 and heat shock techniques

3. How do you make Bacteria competent?
Step 1: Add Calcium Chloride (CaCl2)
CaCl2 is in a solution (creates Ca+2 and Cl- ions)
DNA in plasmid is negatively charged due to phosphate groups in the backbone
Cell membrane of E. coli also is negatively charged because phospholipids are made of same phosphate groups (PO4-3)
Ca+2 ions neutralize charges so plasmid can get near (and inside) bacterial cell.

4. How do you make Bacteria competent?
Step 1: Use Heat Shock
Heat Shock is a process that uses warm water (bath) and ice to help get plasmid inside cell
Add recombinant plasmid + host cell + CaCl2 solution to ice then heat then back on ice
Heat = increases kinetic energy of matter
Molecules/atoms move faster
Ice = decreases kinetic energy of matter
Molecules/atoms move slower

5. 2 types of Bacterial Growth in this lab
Colony growth
Lawn growth

6. GFP

7. Step 1: DNA Isolation  digesting the ”gene of interest” with restriction enzyme
In the lab, this has been done for you!
Gene of Interest = GFP

8. After Isolating GTP from a jellyfish … Step 2: Make Recombinant DNA
Amp

9. RECALL WHAT THE PLASMID (pGLO) LOOKS LIKE…
Step 3 Transformation
RECALL WHAT THE PLASMID (pGLO) LOOKS LIKE…
CaCl2

10. Step 3 Transformation Bacteria is ALSO “Ampicillin Resistance” pGLO
GFP protein
Bacteria is ALSO
“Ampicillin
Resistance”
Amp resistance
pGLO

11. Transformed Bacteria!
When will this happen?

12. What is the ARA operon?
-Clusters of genes located together and transcribed from ONE promoter.
-Ara operon = Inducible operon
-3 arabinose structural genes present natural (not recombined) plasmid: araB, araA, araD
-All 3 genes dependent on promoter (pBAD)
-Arabinose (sugar) changes the shape of the promoter (INACTIVATES REPRESSOR)
INDUCES transcription by allowing RNA polymerase to bind to the DNA

13. = ARABINOSE (needed to make room for RNA polymerase)
Visualize the Operon
Promoter called Pbad
araB
araD
araA
Repressor
= ARABINOSE (needed to make room for RNA polymerase)
Repressor
Pbad
araA
araB
RNA
Polymerase
araD

14. Visualize the pGLO recombinant DNA
What was changed?
Pbad
araB
araD
araA
RNA
Polymerase
Repressor
Pbad
GFP gene
RNA
Polymerase

15. Some of your Materials

16. Lab Procedure-Brief Overview
Label micro test tubes (+pGLO and –pGLO)
Transfer 250 μL (0.25 mL) of transformation solution (CaCl2) to each tube  place on ice
Transformation Solution (CaCl2)

17. Lab Procedure-Brief Overview
3. Use sterile inoculating loop to transfer ~2 “fat” colonies of bacteria to +pGLO tube  spin loop to remove bacteria from loop to CaCl2 solution
4. Use a DIFFERENT sterile inoculating loop to transfer ~2 “fat” colonies of bacteria to -pGLO tube
DO NOT GET TOO MUCH BACTERIA
NO chunks!

18. Lab Procedure-Brief Overview
5. You will need pGLO plasmid…
Your TEACHER will micropipette 10 μL of plasmid into your +pGLO tube
Mix plasmid into the cell suspension of the by tapping the CLOSED microtube on your desk!
Return the tube it to on ice.
DO NOT add plasmid DNA to the –pGLO tube.

19. Lab Procedure-Brief Overview

20. Lab Procedure-Brief Overview
6. Incubate both +pGLO and –pGLO tubes on ice for 10 minutes
-pGLO
+pGLO
10:00

21. Lab Procedure-Brief Overview
While the tubes are sitting on ice…
7. Label your 4 LB Nutrient agar plates on the bottom (not the lid) as follows:       
Label one LB/amp plate: + pGLO      
Label the LB/amp/ara plate: + pGLO       
Label the other LB/amp plate: - pGLO     
Label the LB plate: -pGLO

22. Lab Procedure-Brief Overview
TIME TO HEAT SHOCK…
8. Use foam rack as a holder, transfer both the +pGLO and -pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds.
Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.
When the 50 seconds are done, RAPIDLY place both tubes back on ice. Incubate tubes on ice for 2 minutes

24. Lab Procedure-Brief Overview
9. Remove the rack with tubes from ice and place on lab bench.
Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to EACH tube and reclose it.
Use a new sterile pipet for the other tube. Incubate tubes for 10 minutes at room temperature.
LB = FOOD

25. Lab Procedure-Brief Overview
10. After 10 min have passed, tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 µl  of liquid onto the appropriate LB agar plates

26. Lab Procedure-Brief Overview
11. Use a new sterile loop for each plate. Spread liquid evenly around surface of LB agar using streaking method.
DO NOT PRESS TOO DEEP INTO THE AGAR.

27. Streaking Plates with bacteria

28. Put all plates in the 37°C incubator upside down for 24 hours.
Lab Procedure
12. Stack up your plates and tape them together. Put your group name and class period on the tape and place the stack of plates upside down
Put all plates in the 37°C incubator upside down for 24 hours.

29. Petri Dish Label LB/amp LB/amp/ara LB
What do this dish have on it?
Hypothesis: Will the bacteria grow on the dish? Y or N
Hypothesis: Will the bacteria GLOW green on the dish? Y or N
+pGLO
LB/amp
LB/amp/ara
-pGLO LB

30. Petri Dish Label LB/amp LB/amp/ara LB
What do this dish have on it?
Hypothesis: Will the bacteria grow on the dish? Y or N
Hypothesis: Will the bacteria GLOW green on the dish? Y or N
+pGLO
LB/amp
Plasmid (with pGLO & AMPR), Luria Broth (Agar), ampicillin
YES-colony
growth NO
LB/amp/ara
Plasmid, Luria Broth, ampicillin, arabonose
Yes-
Colony growth
Yes
-pGLO
No plasmid, Luria Broth, ampicillin No LB
No plasmid, Luria broth
Yes- Lawn growth
NEGATIVE CONTROL
POSITIVE CONTROL

31. Results
+ control
- control