Bacterial TransformationAP Biology/Honors Genetics

Ms. Gaynor

What does it mean to transform a cell?

How would a living organism be transformed?

To insert a foreign piece of DNA into a cell

Using a cloning vector/ plasmid

DNA RNA Protein Trait

Don’t Forget…

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 TransformationRECALL WHAT THE PLASMID (pGLO)

LOOKS LIKE…

CaCl2

Step 3 Transformation

pGLO

Amp 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

araB araDaraA

= ARABINOSE (a sugar needed to make room

for RNA polymerase)

Promoter called Pbad

araAaraBRNA

Polymerase

araDPbad

Repressor

Repressor

Visualize the pGLO recombinant DNA

What was changed?araB araDaraAPbad

GFP gene RNA

Polymerase

amp genePbad

Repressor

RNA

Polymerase

Also on the pGLO plasmid

but NOT in place of the ara operon!

Pamp

pGLO plasmid contains:1)ampR gene2)GFP gene 3)Arabinose operon and promoter (pBAD)

operonpromoter

Materials

Lab Procedure-Brief Overview1. 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 Overview3. 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 OverviewLAB 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 Overview6. Incubate both +pGLO and –pGLO tubes on ice

for 10 minutes

+p

GL

O+

pG

LO

-pG

LO

-pG

LO10:0

0

Lab Procedure-Brief OverviewWhile 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 OverviewTIME 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 Overview9. 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 Overview9. 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 Overview10. 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 Overview11. 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 Procedure12. 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•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

Petri Dish Label

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+pGLO

LB/amp/ara-pGLO

LB/amp-pGLO

LB

Petri Dish Label

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

+pGLO

LB/amp/ara

Plasmid, Luria Broth,

ampicillin, arabonose

Yes-

Colony growth Yes

-pGLO

LB/amp

No plasmid, Luria Broth, ampicillin

No No

-pGLO

LB

No plasmid, Luria broth

Yes- Lawn growth No

NEGATIVE CONTROL

POSITIVE CONTROL

Results

+ control - control