Download Bacterial Transformation

BRIDGES 2014
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DNA➔RNA➔PROTEIN➔TRAIT
Genotype
Phenotype
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Adds new information to DNA
Cell can take up (take inside) and expresses a
new piece of DNA
◦ Transformation
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New genetic information often provides
organism with new trait-identifiable after
transformation
Genes can be cut out of human, animal, or
plant DNA and placed inside bacteria via
transformation
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Agriculture
◦ Frost, pest, or drought resistance
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Bioremediation
◦ Bacteria to digest oil spills
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Medicine
◦ Transforming a sick person’s cells with healthy
copies of the defective gene
◦ Creating insulin for diabetes
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Cells that take up DNA
Cells in log phase
◦ Growing cells take up DNA better
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Transformation solution-CaCl2
◦ Ca2+ cation neutralizes the repulsive negative
charges of the phosphate backbone of the DNA and
the phospholipids of the cell membrane
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Heat shock
◦ Increases the permeability of the cell membrane
◦ Duration of the heat shock is critical
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Vehicles for foreign DNA
Plasmid DNA usually contains genes for one
or more traits that may be beneficial to
bacterial survival
Bacteria can transfer plasmids back and forth,
allowing them to share beneficial genes
Allows for adaptation to new environments
Recent occurrence of bacterial antibiotic
resistance due to transmission of plasmids
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Competent cells take up DNA
DNA is brought in on plasmids
Process is called transformation
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GFP gene from jellyfish- Aequorea victoria
◦ Causes cells to glow green under ultraviolet light
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Mutations to GFP enhance fluorescence
Modified GFP gene in pGLO plasmid
After transformation bacteria express the
jellyfish gene and glow bright green
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Gene for GFP
◦ Switched on by adding the sugar arabinose to the
cell’s nutrient medium
◦ Transformed cells are white on plates not
containing arabinose
◦ Transformed cells glow green when arabinose is
included in the nutrient agar
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Gene for resistance to the antibiotic ampicillin
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ori- origin
bla- ampicilin
resistance
araC- activated by
arabinose, allows
down stream
transcription
GFP- green
fluorescent protein
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Add E. coli grown overnight to transformation
solution
◦ Should be in log phase
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Add pGLO plasmid to +pGLO sample
Heat
◦ Should make cells competent (take up plasmid)
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Add to LB plates with ampicillin and
arabinose
Grow overnight
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Label tubes
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Initials
Group number
+pGLO
-pGLO
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Add 250µL transformation solution
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Put on ice
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Add E. coli grown overnight to both tubes
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Add pGLO plasmid to +pGLO tube
DO NOT ADD TO –pGLO TUBE
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Add pGLO plasmid to +pGLO tube
DO NOT ADD TO –pGLO TUBE
Use pipet to transfer 10µL of plasmid
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Put back on ice for 10 mins
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Label plates
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Heat Shock
◦ Time this!
◦ Then ice for 2 mins
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Add broth
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Add broth
Use broth YOU made
Using sterile technique pour some into
beaker and transfer from there
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Add broth
Longer room temperature incubation will
improve results
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Add to plates
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Spread plates
◦ Use different spreaders for each plate
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Incubate overnight
◦ Label with initials
◦ At 37˚C