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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).
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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) 3. Green Fluorescent Protein (GFP) “Tagging”
GFP Excitation Wavelength (e.g. 490 nm) Emission Wavelength (e.g. 510 nm)
Gene Expression DNA (Gene X) Transcription mRNA Translation Protein X
Transcription Translation GFP Tagging Approach DNA (Gene X -GFP “Fusion”) mRNA Protein X-GFP “Fusion”
GFP Tagging Experiments Nuclei Mitotic Spindle Tubulin-GFP Histone-GFP
Question: Where is the Cdc10 protein located in a yeast cell?
Saccharomyces cerevisiae (Yeast) Eukaryotic cell 15 million bp DNA ~ 6000 genes Complete genome sequence known!
Transcription Translation GFP Tagging Approach DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”
Project Overview Isolation of CDC10 gene Open Reading Frame Purification of Genomic DNA from yeast Polymerase Chain Reaction (PCR) Construction of CDC10-GFP “fusion” gene Restriction endonuclease/Ligase Cloning DNA in E. coli Introduction of CDC10-GFP “fusion” gene into yeast cells Observe Cdc10 protein localization in living cells with fluorescence microscopy
Transcription Translation GFP Tagging Approach DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”
Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350
Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350
CDC10 Gene Primers CDC10-For 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Rev 5’ – GTGGTGAAGCTTCTAGCAGCAGCAGTACCTGT – 3’
First Cycle of PCR 5’ 3’ Rev CDC10 3’ 5’ 5’ 3’ (52o C.) (94o C.) (72o C.) For 5’ 3’ Pg. 349
Three Cycles of PCR Pg. 349
Three Cycles of PCR Pg. 349
Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350
CDC10 Gene Sequence
Lab #1 Purify genomic DNA 15 million bp ~ 6500 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350
+ Wells 2000 bp 500 bp
+ Wells 2000 bp 500 bp
3’ 3’ 5’ 5’ 5’ 3’ 5’ 3’ Restriction Endonuclease Reaction 3’ 5’ 3’ 5’ HindIII (37o C.)
DNA Ligase + ATP (15o C.) 3’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ HindIII recognition site is reconstituted Ligation Reaction “Compatible” ends 1. Annealing 2. Phosphodiester bond formation
Construction of a Recombinant DNA Plasmid (insert) Pg. 344
CDC10 Gene Primers CDC10-For 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Rev 5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’
GTGGTGAAGCTTATGTCCATCGAAGAA CACCACTTCGAATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAAAGCTTCACCAC TGACGACGACGATCTTTCGAAGTGGTG 3’ 3’ 5’ CDC10 ORF DNA from PCR
GTGGTGAAGCTTATGTCCATCGAAGAA CACCACTTCGAATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAAAGCTTCACCAC TGACGACGACGATCTTTCGAAGTGGTG 3’ 3’ 5’ AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ CDC10 ORF DNA from PCR HindIII
Ori AmpR pGFP Plasmid HindIII
ACT1p CDC10 orf GFP orf HindIII HindIII - Thr - Ala - Ala - Ala - Arg - Lys - Leu - Met - Ser - Lys - Gly - Cdc10 GFP pCDC10-GFP Plasmid HindIII Site ACT GCT GCT GCT AGA AAG CTTATG TCT AAA GGT 5’ 3’
Ori AmpR pGFP Plasmid AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ HindIII CDC10 orf
Construction of a Recombinant DNA Plasmid (insert) Pg. 344
Transformation of E. Coli plasmid
Transformation of E. Coli Cold CaCl2 plasmid
DNA Cloning Bacterial Transformation (Lab #4) pCDC10-GFP Plasmid Purification (Lab #5) (AmpR) (Ampicillin sensitive) (LB growth medium with ampicillin) Pg. 344
DNA Cloning Bacterial Transformation (Lab #4) pCDC10-GFP Plasmid Purification (Lab #5) (AmpR) (Ampicillin sensitive) (LB growth medium with ampicillin) Pg. 344
DNA Cloning Plasmid Purification (Lab #5) (AmpR) (LB growth medium with ampicillin) Pg. 344
DNA Cloning pCDC10-GFP (LB-amp) (AmpR) (Ampicillin sensitive) (LB-amp Plate) Pg. 344
E. coli Cell Wall Cell Membrane Cytoplasm (chromosome, plasmids)