Biorad%20pGlo-Biotechnology

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Biorad pGlo-Biotechnology
Biotechnology
Explorer Program
Serious About Science Education
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Molecular Biology Lab 6
Bacterial Transformation p GLO
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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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Green Fluorescent Protein
Aequorea victoria
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What makes jelly fish glow?
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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Alba the GFP Bunny
Alba the GFP Bunny
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What is transformation?

• Uptake of foreign DNA, often a circular plasmid

GFP
Amp Resistance
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What is a plasmid?

• A circular piece of autonomously replicating DNA

• Originally evolved by bacteria
• May express antibiotic resistance gene or
• be modified to express proteins of interest

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pGlo plasmid

• Beta Lactamase
• 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

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Bacterial Transformation
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Bacterial DNA
Genomic
Bacterial cell
Plasmid DNA
Genomic DNA
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Transcription regulation

• Lactose operon
• Arabinose operon
• pGLO plasmid

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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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ARABINOSE OPERON
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Expression of Green Fluorescent Protein
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Ara Operon and ara GFP Operon
ara Operon
araC
B
A
D
Effector (Arabinose)
Effector (Arabinose)
araC
B
A
D
RNA Polymerase
B
A
D
araC
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lac Operon and ara operon
RNA Polymerase
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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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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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Reasons for performing each transformation step

• Transformation solution CaCl2
• pH 6.1

Positive charge of Ca2 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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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

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What is nutrient broth or LB broth?
What is nutrient 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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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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Which colonies will glow?
Grow? Glow?

• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?

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How do I measure 250 micro liters using the
pipette?
Volume Measurement
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Central Framework of Molecular Biology