Bacterial Transformation with (pGLO Plasmid)

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Bacterial Transformation with (pGLO Plasmid)

• Lab 10 Molecular Biology

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Purpose of this Lab

• Learn how to insert a gene into bacteria
• (Heat Shock)
• Analyze how a gene can transform an organism and
  express that gene
• Provide evidence that bacteria can take in
  foreign DNA in the form of a plasmid
• Reinforce the following process
• DNA ? RNA ? Protein ? Trait
• Observe how genes are regulated

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Applications of Genetic Transformation

• Used in many areas of Biotechnology
• Agriculture (pests, frost, drought)
• Bacteria (oil spills)
• Gene therapy (sick cells into healthy cells)
• Medicine (produce insulin hormones)

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Key Terms to Know

• DNA Plasmid
• Bacteria E. coli (strain HB101K-12)
• Growth media LB Broth (Luria Bertani)
• Ampicillin Antibiotic kills bacteria amp
• Arabinose Sugar source for energy carbon
• Heat shock Process that increases permeability
  of the cell membrane to DNA
• GFP Green Fluorescent Protein (w/UV)

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The Genes of Interest

• Ampicillin resistance
• Gene regulation proteins-activate the GFP gene
  when arabinose is present
• GFP Green Fluorescent Protein
• -originally isolated from the jellyfish
• Aequorea victoria

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Basic Process

• -Begin with Starter colonies of E. coli
• -Place a colony into each of the two tubes
  provided.
• labeled -pGlo and pGlo
  (yellow loop)
• -Add the pGlo plasmid to the pGlo tube only
  (yellow loop)
• -Place tubes on ice (10 min)
• bottom of tubes should be exposed to the
  ice
• -Label the four plates as indicated in your guide
• -Heat Shock the tubes in water bath (50 sec.)
• -Return to the ice (2 min)
• -Add 250 ?L of LB broth to both tubes (sterile
  pipet each time)
• -Add 100 ?L of tube contents to the approriate
  plates
• -Spread the samples (w yellow loop) tape all
  four plates together. Be sure your period and
  group name is clearly indicated.

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The Process of Heat Shock

• Helps to increase the bacterial uptake of foreign
  DNA
• Membrane becomes more permeable to DNA
• Time is essential
• -ice ? water bath (42ºC) for 50 sec.? ice
• The number of transformants should increase

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Adding Transformation Solution
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Using the Pipet Properly

• Find the markings for each graduated volume on
  the pipet

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Transferring Colonies, Labeling the Plates,
Using Heat Shock
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Adding the Bacteria to the labeled Plates
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Expected Results
PLATES OBSERVATIONS
pGlo LB/amp Many colonies with white appearance Transformation observed (resistance to amp) NO fluorescence (No arabinose present)
pGlo LB/amp/ara Many transformed white colonies Fluoresce bright green under UV light
-pGlo LB/amp (CONTROL) No Bacterial growth present on the plate No transformation
-pGlo LB only (CONTROL Bacteria present with whitish colonies (regeneration of the starter plate)