Bacterial Transformation
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Transcript of Bacterial Transformation
Bacterial Transformation
BRIDGES 2014
DNA➔RNA➔PROTEIN➔TRAIT
Central Dogma
GenotypePhenotype
Adds new information to DNA Cell can take up (take inside) and expresses
a new piece of DNA◦ Transformation
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
Transformation
Agriculture◦ Frost, pest, or drought resistance
Bioremediation◦ Bacteria to digest oil spills
Medicine◦ Transforming a sick person’s cells with healthy
copies of the defective gene◦ Creating insulin for diabetes
Transformation Uses
Cells that take up DNA Cells in log phase
◦ Growing cells take up DNA better Transformation solution-CaCl2
◦ Ca2+ cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane
Heat shock ◦ Increases the permeability of the cell membrane◦ Duration of the heat shock is critical
Competent Cells
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
Plasmids
Competent cells take up DNA DNA is brought in on plasmids Process is called transformation
Overview
GFP gene from jellyfish- Aequorea victoria◦ Causes cells to glow green under ultraviolet light
Mutations to GFP enhance fluorescence Modified GFP gene in pGLO plasmid After transformation bacteria express the
jellyfish gene and glow bright green
Green Fluorescent Protein (GFP)
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 Gene for resistance to the antibiotic
ampicillin
pGLO Plasmid
ori- origin bla- ampicilin
resistance araC- activated by
arabinose, allows down stream transcription
GFP- green fluorescent protein
pGLO Plasmid
Add E. coli grown overnight to transformation solution ◦ Should be in log phase
Add pGLO plasmid to +pGLO sample Heat
◦ Should make cells competent (take up plasmid) Add to LB plates with ampicillin and
arabinose Grow overnight
Activity
Label tubes◦ Initials◦ Group number◦ +pGLO◦ -pGLO
Step 1
Add 250µL transformation solution
Step 2
Put on ice
Step 3
Add E. coli grown overnight to both tubes
Step 4
Add pGLO plasmid to +pGLO tube DO NOT ADD TO –pGLO TUBE
Step 5
Add pGLO plasmid to +pGLO tube DO NOT ADD TO –pGLO TUBE Use pipet to transfer 10µL of plasmid
Step 5 MODIFIED
Put back on ice for 10 mins
Step 6
Label plates
Step 7
Heat Shock◦ Time this!◦ Then ice for 2 mins
Step 8
Add broth
Step 9
Add broth Use broth YOU made Using sterile technique pour some into
beaker and transfer from there
Step 9 MODIFIED
Add broth Longer room temperature incubation will
improve results
Step 9 MODIFIED
Step 10 Add to plates
Spread plates◦ Use different spreaders for each plate
Step 11
Incubate overnight◦ Label with initials◦ At 37˚C
Step 12