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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 Presentation

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Page 1: Question:
Page 2: Question:

Question:

How do we know where a particular protein is located in the cell?

Page 3: Question:

Principle of Fluorescence

Cell with fluorescent molecule

Page 4: Question:

Experimental Approaches for Protein Localization

1. Small Molecule Dyes (e.g. DAPI)

2. Immunostaining (dye-conjugated antibodies)

3. Green Fluorescent Protein (GFP) “Tagging”

Page 5: Question:

Aequorea victoria

Page 6: Question:

Green Fluorescent Protein (GFP)

Page 7: Question:

ExcitationWavelength(e.g. 490 nm)

EmissionWavelength(e.g. 510 nm)

GFP

Page 8: Question:

Gene Expression

DNA (Gene X)

mRNA

Protein X

Transcription

Translation

Page 9: Question:

GFP Tagging Approach

mRNA

DNA (Gene X -GFP “Fusion”)

Protein X-GFP “Fusion”

Transcription

Translation

Page 10: Question:

GFP Tagging Experiments

Nuclei Mitotic Spindle

Histone-GFP Tubulin-GFP

Page 11: Question:

Question:

Where is the Cdc10 protein located in a yeast cell?

Page 12: Question:

*

Septin Protein Family

Page 13: Question:

GFP Tagging Approach

mRNA

DNA (CDC10 -GFP “Fusion”)

Cdc10-GFP “Fusion”

Transcription

Translation

Page 14: Question:

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

Page 15: Question:

GFP Tagging of Cdc10

mRNA

DNA (CDC10 -GFP “Fusion”)

Cdc10-GFP “Fusion”

Transcription

Translation

Page 16: Question:

Saccharomyces cerevisiae (Yeast)

Eukaryotic cell15 million bp DNA~ 6000 genesComplete genome sequence known!

Page 17: Question:

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

Page 18: Question:

Taq DNAPolymerase

Page 19: Question:

Pg. 202 DNA Synthesis

Primer

Page 20: Question:

CDC10-Forward

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Reverse

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

Page 21: Question:

CDC10 GeneSequence

(non-template strand sequence)

Page 22: Question:

First Cycle of PCR

Pg. 349

(94o C.) (52o C.)(72o C.)

CDC10

For

Rev

5’5’ 3’3’

3’

3’ 5’

5’

Page 23: Question:

Three Cycles of PCR

Pg. 349

Page 24: Question:
Page 25: Question:

Agarose

Gelidium comeum (kelp)

Page 26: Question:

Ethidium Bromide

Page 27: Question:

+

Page 28: Question:

+

Page 29: Question:

Restriction Endonuclease Reaction

HindIII (37o C.)

5’

5’

3’

3’

5’

5’3’

3’3’

3’5’

5’

Page 30: Question:

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

Page 31: Question:

Pg. 344

Construction of a Recombinant DNA Plasmid

(insert)

Page 32: Question:

CDC10-For

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Rev

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

Page 33: Question:

GTGGTGAAGCTTATGTCCATCGAAGAACACCACTTCGAATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAAAGCTTCACCACTGACGACGACGATCTTTCGAAGTGGTG

5’3’ 5’

3’

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 ORF DNA from PCR

HindIII

Page 34: Question:

Ori

AmpR

pGFP Plasmid

HindIII

Page 35: Question:

Ori

AmpR

pGFP Plasmid

HindIII

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 orf

Page 36: Question:

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

Page 37: Question:

Transformation of E. Coli

plasmid

Page 38: Question:

Pg. 344

(Ampicillin sensitive)

(AmpR)

(LB growth medium with ampicillin)

DNA Cloning

pCDC10-GFP

PlasmidPurification (Lab #6)

Bacterial Transformation(Lab #5)

Page 39: Question:

Ori

AmpR

pGFP Plasmid

HindIII

Page 40: Question:

Ampicillin

Inhibits cell wall synthesis

Page 41: Question:

Pg. 344

(Ampicillin sensitive)

(AmpR)

DNA Cloning

pCDC10-GFP

(LB-amp Plate)

(LB-amp)

Page 42: Question:

Transformation of E. Coli

plasmid

Log Phase GrowthCold (4oC)CaCl2