pGLO Transformation LAB AP LAB 6

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pGLO Transformation LABAP LAB 6
BIO-RAD lab book
http//www.mshri.on.ca/nagy/GFP20mice.jpg
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Aequorea victoria Source of glowing gene for
this experiment
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Jellyfish Gene put into Other Critters
http//www.lafuga.de/GFP_pig.jpg
http//www.technologyreview.com/files/21291/monkey
_x600.jpg
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• PLASMID
• Extrachromosomal DNA
• Often carry genes for antibiotic resistance
• Can be passed from one bacterium to another

http//www.agen.ufl.edu/owens/age2062/OnLineBiolo
gy/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK
/14_1.jpg
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Bacterial Transformation
The uptake of DNA
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pGLO LAB SUPPLIES

• FOAM tube holder/float
• 4 - flip top microtubes
• Blue- Transforming solution (CaCl2)
• Yellow- LB nutrient broth
• Pink- label
• Purple- label -
• 1- colored eraser (to ID your tubes in water
  bath)
• 1-pkg yellow innoculating loops
• 2- Sterile pipettes
• 4 poured agar plates
• 1 - LB
• 2 - LB/amp
• 1- LB/amp/ara
• PERMANENT MARKER
• Cup with crushed ice

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• LABEL TUBES

Purple pGLO pink - pGLO
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Transformationsolution (CaCl2)

• Use sterile pipette to
• add 250µL transformation
• solution to pGLO and tubes

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Get your rack on ICE!
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INNOCULATE TUBES WITH E. coli BACTERIA
Pick one colonyTwirl loop in pGLO tube Get
new loopPick one colonyTwirl loop in pGLO tube
USE SPECIAL GARBAGE BAG FORDISPOSAL OF USED
LOOPS
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EXAMINE pGLO plasmid DNA

• Use UV light to examine pGLO plasmid vial
• UV light can be harmful to your eyes!Wear your
  goggles.Do not shine in eyes.
• GFP Green Fluorescent Protein
• isolated from jellyfish
• USED AS A GENETIC TOOL

http//www.mshri.on.ca/nagy/GFP20mice.jpg
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PLASMID DNA TRANSFER

• THIS STEP IS CRUCIAL!
• Look closely to make sure you have a film of
  solution across the ring.
• (Similar to soapy film when you blow bubbles)

ADD PLASMID TO TUBE DO NOT ADD PLASMID TO -
TUBE
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Get your rack on ICE!
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• WHILE YOUR TUBES COOL
• LABEL YOUR PLATES

TIP UPSIDE DOWN AND WRITE LABELS ON BOTTOM
NOT ON TOP!
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• LB (Luria and Bertani) broth agar
• provides nutrients for bacterial growth
• LB/amp
• Luria agar ampicillin (antibiotic)
• LB/amp/ara
• Luria agar ampicillin arabinose sugar

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• SHOCKING INCREASES UPTAKE OF FOREIGN DNA
  (PLASMID)
• OSMOTIC SHOCK Transforming solution
• CaCl2
• HEAT SHOCK RAPID TEMPERATURE CHANGE is the key

50 SECONDS
2 MINUTES
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• Place foam rack with and tubes on desktop
• Use new sterile pipette to add 250 µL Luria
  broth to tube
• Use new sterile pipette to add 250 µL Luria
  broth to tube
• Incubate a ROOM TEMPERATURE 10 min

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TAP WITH FINGER TO MIX! Use NEW STERILEpipette
for each vial to add 100 uL bacterial
suspension to CORRECT DISH (CHECK LABELS!) Use a
NEW STERILELOOP FOR EACH PLATE to spread
suspension evenly on surface of plate DO NOT DIG
INTO AGAR! QUICKLY REPLACE LIDS
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FLIP PLATES UPSIDE DOWNSTACK AND TAPE LABEL
WITH YOUR GROUP NAMEPLACE IN INCUBATOR
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pGLO plasmid
ARABINOSE OPERON (INDUCIBLE) Turns on when
arabinose sugar is present Allows
bacteria to digest this sugar
Ori- Plasmid Replication genes
GFP-Green Fluorescent Protein - Glows green in
fluorescent light
bla (beta-lactamase) - On all time - Makes
protein that breaks down ampicillin - Provides
ampicillin resistance
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ARABINOSE OPERON REGULATION
ara Operon
INDUCIBLE OPERON PRESENCE OF ARABINOSE TURNS ON
GENES TO MAKE ENZYMES TO DIGEST ARABINOSE
araC
B
A
D
Effector (Arabinose)
araC
B
A
D
RNA Polymerase
B
A
D
araC
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pGLO Regulation
ara Operon
GFP GENE HAS BEEN ADDED TO ara OPERON WHEN
ARABINOSE IS PRESENT, OPERON IS TURNED ON
andGFP GENEIS EXPRESSED TOO Cells glow on
mediawith arabinose
araC
B
A
D
GFP Gene
Effector (Arabinose)
GFP Gene
araC
B
A
D
RNA Polymerase
B
A
D
araC
GFP Gene
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Reasons for Each Transformation Step

• CaCl2 treatment
• Positive charge of Ca2 ions neutralizes
• negative charge of DNA phosphates
• negative charge of membrane phospholipids

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Reasons for Each Transformation Step

• Incubation on ice slows fluid cell membranes
• Heat-shock increases permeability of cell
  membrane
• Nutrient broth incubation allows beta lactamase
  expression

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Selection for plasmid uptake

• Antibiotic becomes a selecting agent
• only bacteria with the plasmid will grow on
  antibiotic (ampicillin) plate

only transformed bacteria grow
all bacteria grow
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LB/amp plate
LB plate
cloning
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Transformation Results
LB PLATELuria Broth - PGLO NO Plasmid
?
All cells grow since there is no antibiotic on
the plate
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Transformation Results
LB/AMP PLATELuria Broth with antibiotic
- PGLO NO plasmid
?
NO GROWTHCells without plasmid dont have
antibiotic resistance. Cant grow on media with
antibiotic added.
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Transformation Results
LB/AMP PLATELuria Broth with antibiotic
PGLO Plasmid added
?
LAWNCells with plasmid have antibiotic
resistance gene so can grow on media with
antibiotic
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Transformation Results
Cells with pGLO plasmid GROW GLOW-can grow on
media with antibiotic GLOW on media with
arabinose (turns on GFP gene)
LB/AMP/ARA PLATELuria Broth antibiotic
arabinose PGLO
Plasmid added
?