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Question:. How do we know where a particular protein is located in the cell?. Cell with fluorescent molecule. Principle of Fluorescence. Experimental Approaches for Protein Localization. 1. Small Molecule Dyes (e.g. DAPI). 2. Immunostaining (dye-conjugated antibodies). - PowerPoint PPT PresentationTRANSCRIPT
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Question:
How do we know where a particular protein is located in the cell?
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Principle of Fluorescence
Cell with fluorescent molecule
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Experimental Approaches for Protein Localization
1. Small Molecule Dyes (e.g. DAPI)
2. Immunostaining (dye-conjugated antibodies)
3. Green Fluorescent Protein (GFP) “Tagging”
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Aequorea victoria
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Green Fluorescent Protein (GFP)
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ExcitationWavelength(e.g. 490 nm)
EmissionWavelength(e.g. 510 nm)
GFP
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Gene Expression
DNA (Gene X)
mRNA
Protein X
Transcription
Translation
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GFP Tagging Approach
mRNA
DNA (Gene X -GFP “Fusion”)
Protein X-GFP “Fusion”
Transcription
Translation
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GFP Tagging Experiments
Nuclei Mitotic Spindle
Histone-GFP Tubulin-GFP
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Question:
Where is the Cdc10 protein located in a yeast cell?
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*
Septin Protein Family
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GFP Tagging Approach
mRNA
DNA (CDC10 -GFP “Fusion”)
Cdc10-GFP “Fusion”
Transcription
Translation
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Project OverviewIsolation of CDC10 gene Open Reading Frame
Purification of Genomic DNA from yeastPolymerase Chain Reaction (PCR)
Construction of CDC10-GFP “fusion” gene
Restriction endonuclease/LigaseCloning DNA in E. coli
Introduction of CDC10-GFP “fusion” gene
into yeast cells
Observe Cdc10 protein localization in living cells with fluorescence microscopy
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GFP Tagging of Cdc10
mRNA
DNA (CDC10 -GFP “Fusion”)
Cdc10-GFP “Fusion”
Transcription
Translation
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Saccharomyces cerevisiae (Yeast)
Eukaryotic cell15 million bp DNA~ 6000 genesComplete genome sequence known!
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Copies of CDC10 Gene Open Reading FramePg. 350
Purify genomic DNA
~ 6000 genes
Lab #1 & 2
15 million bp
PCR
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Taq DNAPolymerase
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Pg. 202 DNA Synthesis
Primer
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CDC10-Forward
5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’
CDC10-Reverse
5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’
CDC10 Gene Primers
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CDC10 GeneSequence
(non-template strand sequence)
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First Cycle of PCR
Pg. 349
(94o C.) (52o C.)(72o C.)
CDC10
For
Rev
5’5’ 3’3’
3’
3’ 5’
5’
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Three Cycles of PCR
Pg. 349
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Agarose
Gelidium comeum (kelp)
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Ethidium Bromide
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+
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+
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Restriction Endonuclease Reaction
HindIII (37o C.)
5’
5’
3’
3’
5’
5’3’
3’3’
3’5’
5’
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Ligation Reaction
“Compatible” ends
DNA Ligase + ATP (15o C.)
HindIII recognition site is reconstituted
5’
5’3’
3’3’
3’5’
5’
3’
3’
5’
5’
1. Annealing
2. Phosphodiesterbond formation
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Pg. 344
Construction of a Recombinant DNA Plasmid
(insert)
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CDC10-For
5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’
CDC10-Rev
5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’
CDC10 Gene Primers
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GTGGTGAAGCTTATGTCCATCGAAGAACACCACTTCGAATACAGGTAGCTTCTT
ACTGCTGCTGCTAGAAAGCTTCACCACTGACGACGACGATCTTTCGAAGTGGTG
5’3’ 5’
3’
AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT
ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA
5’3’ 5’
3’
CDC10 ORF DNA from PCR
HindIII
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Ori
AmpR
pGFP Plasmid
HindIII
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Ori
AmpR
pGFP Plasmid
HindIII
AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT
ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA
5’3’ 5’
3’
CDC10 orf
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ACT GCT GCT GCT AGA AAG CTT ATG TCT AAA GGTHindIII Site
- Thr - Ala - Ala - Ala - Arg - Lys - Leu - Met - Ser - Lys - Gly -
Cdc10 GFP
5’ 3’
pCDC10-GFP Plasmid
CDC10 orf GFP orfACT1pHindIII HindIII
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Transformation of E. Coli
plasmid
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Pg. 344
(Ampicillin sensitive)
(AmpR)
(LB growth medium with ampicillin)
DNA Cloning
pCDC10-GFP
PlasmidPurification (Lab #6)
Bacterial Transformation(Lab #5)
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Ori
AmpR
pGFP Plasmid
HindIII
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Ampicillin
Inhibits cell wall synthesis
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Pg. 344
(Ampicillin sensitive)
(AmpR)
DNA Cloning
pCDC10-GFP
(LB-amp Plate)
(LB-amp)
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Transformation of E. Coli
plasmid
Log Phase GrowthCold (4oC)CaCl2