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

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

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