1. Bacterial Transformation
AP Biology/Honors Genetics
Ms. Gaynor

2. What does it mean to transform a cell?
To insert a foreign piece of DNA into a cell
How would a living organism be transformed?
Using a cloning vector/ plasmid

3. Give it different DNA

4. Don’t Forget…
DNA
RNA
Protein
Trait

5. Bacterial Transformation
Step 1 DNA Isolation
Isolation of Your Gene of Interest (GFP)
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
Use CaCl2 and heat shock techniques

6. Step 1: DNA Isolation  digesting the ”gene of interest” with restriction enzyme
In the lab, this has been done for you!
How did they do it?

7. After Isolating GFP from a jellyfish … Step 2: Make Recombinant DNA
Amp

8. Need to use Restriction Enzymes to Make Recombinant DNA
Act as molecular scissors
Naturally found in bacteria
Used to decompose viral & phage DNA
Act as ENDOnucleases (cut WITHIN the DNA strand)
~3,000 known enzymes

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 an operon?
-Clusters of genes located together and transcribed from ONE promoter  usually found only in bacteria
-3 arabinose genes are present in a natural (not recombined) plasmid: araB, araA, araD
-All 3 genes dependent on 1 promoter (called pBAD)
-Interaction with arabinose (sugar) changes the shape of the promoter & enables RNA polymerase to bind to the DNA coding strand for transcription

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

15. Visualize the pGLO recombinant DNA
What was changed?
Pbad
araB
araD
araA
RNA
Polymerase
Also on the pGLO plasmid but NOT in place of the ara operon!
Repressor
Pbad
Pamp
GFP gene
RNA
Polymerase
amp gene

16. pGLO plasmid contains:
ampR gene
GFP gene
Arabinose operon and promoter (pBAD)
operon
promoter

17. Materials

18. 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)

19. 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
NO chunks!

20. Lab Procedure-Brief Overview
LAB PRCEDURE REVISION
5. I will have some pGLO plasmid in a labeled micro test tube…
You (a lab group member) will come to the front of the room and retrieve your 9 μL of pGLO plasmid
I will add plasmid using a micropipette 
Cap the +pGLO microtest tube and mix the plasmid into the cell suspension by inverting tube.
Return this test tube to the ice.
DO NOT add plasmid DNA to the –pGLO tube.
You are NOT doing this method!!!

21. Lab Procedure-Brief Overview

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

23. 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

24. 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

26. 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.

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

28. 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

29. 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.

30. Streaking Plates with bacteria

31. 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 in Ms. Gaynor’s transfer box.
She will put all plates in the 37°C incubator upside down for 24 hours.

32. Reasons for Performing Transformation Step
1. Transformation solution = CaCI2
-Positive charge of Ca++ ions shields negative charge of DNA phosphates & helps neutralize cell membrane so plasmid can get in
2. Incubate on ice
-Slows movement of cell membrane so Ca++ can bind & plasmid can slip into bacterial cell
3. Heat-shock
-Increases movement of membranes (heat)
- Then closes up holes in membranes
4. Nutrient broth incubation
-Allows bacteria to be feed 

33. Review… What does the following mean?
+pGLO
a cell contains the pGLO plasmid (transformed cell)
pGLO plasmid contains 2 genes: AMP and GTP
-pGLO
a cell without the plasmid (“normal” cell) LB
Luria Broth (LB or Agar)  sugar needed for E.Coli to live (feeds on this sugar solution)
amp
Ampicillin  an liquid antibiotic added to the LB
Normally KILLS bacteria by breaking down cell wall peptidoglycan
ara
Arabinose  sugar needed to turn on operon containing the GFP gene; needed to make glow protein

34. 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

35. Petri Dish Label LB/amp LB/amp/ara LB
What do this dish have on it?
**they all have E. coli
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

36. Results
+ control
- control