gfp expression overview aug2010€¦ · green fluorescent protein • normally found in the rim of...
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GFP Expression
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Green Fluorescent Protein
• Normally found in the rim of Jellyfish
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jellyfish produces green bioluminescencefrom small photoorgans located on its
umbrella
• When the rings of twentyto thirty jellyfish aresqueezed through arayon gauze, a faintlyluminescent liquid called“squeezate” is obtained
• GFP Green Fluorescentprotein can extractedfrom “Squeezate”
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Our system
• GFP gene is on a plasmid (small DNA molecule)with the selectable marker of Amp and under aninducible Arabinose promoter.
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What is a plasmid?• A circular piece of
autonomouslyreplicating DNA
• Originally evolved bybacteria
• May express antibioticresistance gene
or be modified toexpress proteins ofinterest
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The Many Faces of Plasmids
Transmission electron micrograph
Agarose gelGraphic representation
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BLA= beta lactamase• BLA encodes for an enzyme Beta
lactamase• The function is to cleave lactam group off
Beta lactams ( penicillin , ampicillin andrelated molecules) to render it in active
• Confers antibiotic resistance• Used as a means of selecting for
tranformed cells
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Transcriptional Regulation
• pGLO plasmid• Arabinose
operon• BLA
Ampicillinase
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Transcriptional Regulation
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
Z Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
Arabinose gene control is similar to Lac operon
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Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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Example of an induction
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Expression of GFP can be linkedwith another gene
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E.Coli cell is like a bag containing allsorts of proteins
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Strategy for GFP purification
• Harvest cells• Lyse cells• Precipitate protein to reduce volume• Apply to chromatography columns;
selectively desorb
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Lysozyme cuts bonds betweenSugars on the cell walls
Cell Wall weakens and the cellbursts open
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Lysozymefacts:
• Lysoszyme is an enzyme that fightsinfections (kills certain types ofbacteria)
• Found in Tears, Saliva and NasalDrippings
• Also found in Chicken egg whites
• Used to prevent Bacterialcontamination in Beer making
• Cows secrete lysozyme into theirstomaches as a digestive enzyme
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Lysozyme in action
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Why Use Chromatography?
• To purify a singlerecombinantprotein of interestfrom over 4,000naturally occuringE. coli geneproducts.
After Lysis A mixture of proteins is released
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Hydrophobic InteractionChromatography:
Steps 1–3Add bacterial lysate to column matrix inhigh salt buffer
2.Wash less hydrophobic proteins fromcolumn in
low salt buffer
3.Elute GFP from column with no salt buffer
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Step 1:Hydrophobic Interaction
Chromatography
• Add bacteriallysate tocolumn matrixin high saltbuffer– Hydrophobic
proteinsinteract withcolumn
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Step 2:Hydrophobic Interaction
Chromatography• Wash less
hydrophobic fromcolumn with lowsalt buffer– Less
hydrophobicE. coliproteins fallfrom column
– GFP remainsbound to thecolumn
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Step 3:Hydrophobic Interaction
Chromatography• Elute GFP from
column byadding no saltbuffer
GFP– Released
from columnmatrix
– Flows throughthe column