Transcript Bacterial Transformation - Baldwinsville Central School

Bacterial
Transformation
AP Biology/Honors Genetics
Ms. Gaynor
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
Don’t Forget…
DNA
RNA
Protein
Trait
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
• http://www.dnalc.org/resources/animations/transformation1.html
• Step 3 Transformation
– Insertion of recombinant DNA into
bacteria by making bacteria competent
• Use CaCl2 and heat shock techniques
• http://www.dnalc.org/resources/animations/transformation2.html
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?
After Isolating GFP from a jellyfish
… Step 2: Make Recombinant
DNA
Amp
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
Step 3 Transformation
RECALL WHAT THE PLASMID
(pGLO) LOOKS LIKE…
CaCl2
Step 3 Transformation
GFP protein
Amp
resistance
Bacteria is ALSO
pGLO
“Ampicillin
Resistance”
Transformed Bacteria!
When will this happen?
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
Visualize the Operon
Promoter
called Pbad
Repressor
araB
Pbad
Polymerase
araD
= ARABINOSE
(a sugar needed
to make room for
RNA polymerase)
Repressor
RNA
araA
araB
araA
araD
Visualize the pGLO recombinant DNA
What was changed?
Pbad
RNA
Polymerase
araB ara
A
araD
Also on the
pGLO
plasmid but
NOT in place
of the ara
operon!
Repressor
Pbad
RNA
Polymerase
GFP gene
Pamp
amp
gene
pGLO plasmid contains:
1)ampR gene
2)GFP gene
3)Arabinose operon and promoter (pBAD)
operon
promoter
Materials
Lab Procedure-Brief Overview
1. Label micro test tubes (+pGLO and –pGLO)
2. Transfer 250 μL (0.25 mL) of transformation
solution (CaCl2) to each tube  place on ice
Transformation
Solution (CaCl2)
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!
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!!!
Lab Procedure-Brief Overview
Lab Procedure-Brief Overview
-pGLO
10:00
+pGLO
6. Incubate both +pGLO and –pGLO tubes on ice
for 10 minutes
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
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
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.
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.
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
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.
Streaking Plates with
bacteria
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.
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 
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
•ara
•Normally KILLS bacteria by breaking down cell
wall peptidoglycan
•Arabinose  sugar needed to turn on operon containing
the GFP gene; needed to make glow protein
What do this
Hypothesis: Will Hypothesis: Will
Petri Dish
dish have on it? the bacteria
the bacteria
Label
+pGLO
LB/amp
+pGLO
LB/amp/ara
-pGLO
LB/amp
-pGLO
LB
grow on the
dish? Y or N
GLOW green on
the dish? Y or N
What do this
Hypothesis: Will Hypothesis: Will
Petri Dish
dish have on it? the bacteria
the bacteria
Label
**they all have
E. coli
+pGLO
LB/amp
+pGLO
LB/amp/ara
-pGLO
LB/amp
-pGLO
LB
grow on the
dish? Y or N
GLOW green on
the dish? Y or N
pGLO &
AMPR), Luria
Broth (Agar),
ampicillin
YES- colony
growth
NO
Plasmid,
Luria Broth,
ampicillin,
arabonose
YesColony
growth
Yes
No plasmid,
Luria Broth,
ampicillin
No
No
Plasmid (with
No plasmid,
Luria broth
NEGATIVE CONTROL
Yes- Lawn
No
growth
POSITIVE CONTROL
Results
+ control
- control