question:

Question:

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

Principle of Fluorescence

Cell with fluorescent molecule

Experimental Approaches for Protein Localization

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

2. Immunostaining (dye-conjugated antibodies)

3. Green Fluorescent Protein (GFP) “Tagging”

Aequorea victoria

Green Fluorescent Protein (GFP)

ExcitationWavelength(e.g. 490 nm)

EmissionWavelength(e.g. 510 nm)

GFP

Gene Expression

DNA (Gene X)

mRNA

Protein X

Transcription

Translation

GFP Tagging Approach

mRNA

DNA (Gene X -GFP “Fusion”)

Protein X-GFP “Fusion”

Transcription

Translation

GFP Tagging Experiments

Nuclei Mitotic Spindle

Histone-GFP Tubulin-GFP

Question:

Where is the Cdc10 protein located in a yeast cell?

*

Septin Protein Family

GFP Tagging Approach

mRNA

DNA (CDC10 -GFP “Fusion”)

Cdc10-GFP “Fusion”

Transcription

Translation

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

GFP Tagging of Cdc10

mRNA

DNA (CDC10 -GFP “Fusion”)

Cdc10-GFP “Fusion”

Transcription

Translation

Saccharomyces cerevisiae (Yeast)

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

Copies of CDC10 Gene Open Reading FramePg. 350

Purify genomic DNA

~ 6000 genes

Lab #1 & 2

15 million bp

PCR

Taq DNAPolymerase

Pg. 202 DNA Synthesis

Primer

CDC10-Forward

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Reverse

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

CDC10 GeneSequence

(non-template strand sequence)

First Cycle of PCR

Pg. 349

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

CDC10

For

Rev

5’5’ 3’3’

3’

3’ 5’

5’

Three Cycles of PCR

Pg. 349

Agarose

Gelidium comeum (kelp)

Ethidium Bromide

+

+

Restriction Endonuclease Reaction

HindIII (37o C.)

5’

5’

3’

3’

5’

5’3’

3’3’

3’5’

5’

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

Pg. 344

Construction of a Recombinant DNA Plasmid

(insert)

CDC10-For

5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’

CDC10-Rev

5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

CDC10 Gene Primers

GTGGTGAAGCTTATGTCCATCGAAGAACACCACTTCGAATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAAAGCTTCACCACTGACGACGACGATCTTTCGAAGTGGTG

5’3’ 5’

3’

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 ORF DNA from PCR

HindIII

Ori

AmpR

pGFP Plasmid

HindIII

Ori

AmpR

pGFP Plasmid

HindIII

AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT

ACTGCTGCTGCTAGAATGACGACGACGATCTTTCGA

5’3’ 5’

3’

CDC10 orf

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

Transformation of E. Coli

plasmid

Pg. 344

(Ampicillin sensitive)

(AmpR)

(LB growth medium with ampicillin)

DNA Cloning

pCDC10-GFP

PlasmidPurification (Lab #6)

Bacterial Transformation(Lab #5)

Ori

AmpR

pGFP Plasmid

HindIII

Ampicillin

Inhibits cell wall synthesis

Pg. 344

(Ampicillin sensitive)

(AmpR)

DNA Cloning

pCDC10-GFP

(LB-amp Plate)

(LB-amp)

Transformation of E. Coli

plasmid

Log Phase GrowthCold (4oC)CaCl2