1. Biorad pGlo-Biotechnology
Explorer Program
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Serious About Science Education

2. Bacterial Transformation
Molecular Biology Lab 6
Bacterial Transformation
p GLO
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3. Links to real world study of biological processes
(ex. biosynthesis of proteins)
localization of gene expression
cell movement
cell fate during development
formation of different organs
screen-able marker to identify
transgenic organisms
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4. Green Fluorescent Protein
Aequorea victoria
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5. What makes jelly fish glow?
Calcium: the body's conductor
Why can photo proteins, the flash-bulb proteins that jellies such as Aequorea Victoria turn on to give the outer edge of the creature's bell-shaped body a bluish or greenish glow? The answer centers on calcium.
The jelly's light switch turns on in the presence of calcium ions, and the calcium ion is one of nature's favorite intercellular messengers,
Whenever you tense a muscle or waggle a finger; whenever your heart pounds out a beat, pulsing blood through a tangle of veins and arteries, calcium has prompted the muscle to act, contract or twitch.
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6. Alba the GFP Bunny
Alba the GFP Bunny
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7. What is transformation?
Uptake of foreign DNA, often a circular plasmid
GFP
Amp Resistance
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8. What is a plasmid? A circular piece of autonomously replicating DNA
ori
bla
Originally evolved by bacteria
May express antibiotic resistance gene or
be modified to express proteins of interest
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9. pGlo plasmid Beta Lactamase Green Fluorescent Protein
Ampicillin resistance The beta lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates ampicillin.
Green Fluorescent Protein
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription
pGLO
ori
bla
GFP
araC
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10. Bacterial Transformation
GFP
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Bacterial
chromosomal DNA
Cell wall
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11. Bacterial DNA Genomic Bacterial cell Plasmid DNA Genomic DNA
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Genomic DNA
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12. Transcription regulation
HELWIG
Transcription regulation
Lactose operon
Arabinose operon
pGLO plasmid
pGLO
ori
bla
GFP
araC
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13. ARABINOSE
SOURCE OF ENERGY AND CARBON FOR BACTERIA. FOUND IN PLANTS AND IS NORMALLY USED AS A SOURCE FOOD FOR BACTERIA.
THE BACTERIAL GENES THAT MAKE DIGESTIVE ENZYMES TO BREAK DOWN ARABINOSE FOR FOOD ARE NOT EXPRESSED WHEN ARABINOSE IS ABSENT.
WHEN ARABINOSE IS PRESENT GENES TURN ON. WHEN ABSENT GENES TURN OFF.
ARABINOSE INITIATES TRANSCRIPTION OF THESE GENES BY PROMOTING THE BINDING OF RNA POLYMERASE.
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14. ARABINOSE OPERON
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15. Expression of Green Fluorescent Protein
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16. Ara Operon and ara GFP Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
ara Operon
araC B A D
Effector (Arabinose)
Effector (Arabinose)
araC B A D
RNA Polymerase
araC B A D
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17. lac Operon and ara operonZ Y A
LacI
Effector
(Lactose)
lac Operon B A D
araC
RNA Polymerase
Effector (Arabinose)
ara Operon
RNA Polymerase
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18. Methods of Transformation
Electroporation
Electrical shock makes cell membranes permeable to DNA
Calcium Chloride/Heat Shock
Chemically-competent cells uptake DNA after heat shock
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19. Transformation procedure
Suspend bacterial colonies in Transformation Solution
Add pGLO plasmid DNA
Place tubes on ice
Heat shock at 42oC and place on ice
Incubate with nutrient broth
Streak plates
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20. Reasons for performing each transformation step
Ca++ O
CH2 P
Base OH
Sugar
Transformation solution = CaCl2
pH = 6.1
Positive charge of Ca+2 ions neutralizes negative charge of phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to enter the cells.
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21. Why perform each transformation step?
Incubation on ice slows fluid cell membranes
Heat-shock increases the permeability of cell membrane to DNA. Increase the bacterial uptake of foreign DNA.
Nutrient broth incubation allows beta lactamase expression
GFP
Cell wall
Beta lactamase
(ampicillin resistance)
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22. What is nutrient broth or LB broth?
Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression
carbohydrates
amino acids
nucleotides
salts
vitamins
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23. RECOVERY
Recovery is the 10 minute incubation period after you added the LB which allows the cells to grow and express the ampicillin resistant beta galactamase so the newly transformed cells survive on ampicllin plates.
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24. Which colonies will glow?
Grow? Glow?
Which colonies will glow?
Follow protocol
On which plates will colonies grow?
Which colonies will glow?
LB/Amp
LB/Amp/Ara LB
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25. How do I measure 250 micro liters using the pipette?
Volume Measurement
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26. Central Framework of Molecular Biology
DNA
RNA
Protein
Trait
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