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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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  1. Question: How do we know where a particular protein is located in the cell?

  2. Cell with fluorescent molecule Principle of Fluorescence

  3. Experimental Approaches for Protein Localization 1. Small Molecule Dyes (e.g. DAPI) 2. Immunostaining (dye-conjugated antibodies) 3. Green Fluorescent Protein (GFP) “Tagging”

  4. Aequorea victoria

  5. Green Fluorescent Protein (GFP)

  6. GFP Excitation Wavelength (e.g. 490 nm) Emission Wavelength (e.g. 510 nm)

  7. Gene Expression DNA (Gene X) Transcription mRNA Translation Protein X

  8. Transcription Translation GFP Tagging Approach DNA (Gene X -GFP “Fusion”) mRNA Protein X-GFP “Fusion”

  9. GFP Tagging Experiments Nuclei Mitotic Spindle Tubulin-GFP Histone-GFP

  10. Question: Where is the Cdc10 protein located in a yeast cell?

  11. Septin Protein Family *

  12. Transcription Translation GFP Tagging Approach DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”

  13. 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

  14. Transcription Translation GFP Tagging of Cdc10 DNA (CDC10 -GFP “Fusion”) mRNA Cdc10-GFP “Fusion”

  15. Saccharomyces cerevisiae (Yeast) Eukaryotic cell 15 million bp DNA ~ 6000 genes Complete genome sequence known!

  16. Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350

  17. Taq DNA Polymerase

  18. Primer DNA Synthesis Pg. 202

  19. CDC10 Gene Primers CDC10-Forward 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Reverse 5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

  20. CDC10 Gene Sequence (non-template strand sequence)

  21. First Cycle of PCR 5’ 3’ Rev CDC10 3’ 5’ 5’ 3’ (52o C.) (94o C.) (72o C.) For 5’ 3’ Pg. 349

  22. Three Cycles of PCR Pg. 349

  23. Three Cycles of PCR Pg. 349

  24. Lab #1 & 2 Purify genomic DNA 15 million bp ~ 6000 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350

  25. Agarose Gelidium comeum (kelp)

  26. Ethidium Bromide

  27. Lab #1 Purify genomic DNA 15 million bp ~ 6500 genes PCR Copies of CDC10 Gene Open Reading Frame Pg. 350

  28. +

  29. +

  30. 3’ 3’ 5’ 5’ 5’ 3’ 5’ 3’ Restriction Endonuclease Reaction 3’ 5’ 3’ 5’ HindIII (37o C.)

  31. 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

  32. Construction of a Recombinant DNA Plasmid (insert) Pg. 344

  33. CDC10 Gene Primers CDC10-For 5’ – GTGGTGAAGCTTATGTCCATCGAAGAACCTAG – 3’ CDC10-Rev 5’ – GTGGTGAAGCTTTCTAGCAGCAGCAGTACCTGT – 3’

  34. GTGGTGAAGCTTATGTCCATCGAAGAA CACCACTTCGAATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAAAGCTTCACCAC TGACGACGACGATCTTTCGAAGTGGTG 3’ 3’ 5’ AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ CDC10 ORF DNA from PCR HindIII

  35. Ori AmpR pGFP Plasmid HindIII

  36. Ori AmpR pGFP Plasmid AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ HindIII CDC10 orf

  37. 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’

  38. DNA Cloning Bacterial Transformation (Lab #5) pCDC10-GFP Plasmid Purification (Lab #5) (AmpR) (Ampicillin sensitive) (LB growth medium with ampicillin) Pg. 344

  39. DNA Cloning Plasmid Purification (Lab #6) (AmpR) (LB growth medium with ampicillin) Pg. 344

  40. Ori AmpR pGFP Plasmid AGCTTATGTCCATCGAAGAA ATACAGGTAGCTTCTT 5’ ACTGCTGCTGCTAGAA TGACGACGACGATCTTTCGA 3’ 3’ 5’ HindIII CDC10 orf

  41. Transformation of E. Coli plasmid

  42. Ori AmpR pGFP Plasmid HindIII

  43. DNA Cloning pCDC10-GFP (LB-amp) (AmpR) (Ampicillin sensitive) (LB-amp Plate) Pg. 344

  44. Transformation of E. Coli Log Phase Growth Cold (4oC) CaCl2 plasmid

  45. E. Coli Cell Wall Cell Membrane Cytoplasm (chromosome, plasmids)

  46. Ori AmpR pCDCGFP Plasmid CDC10GFP HindIII HindIII

  47. 1000 bp Morning section

  48. 1000 bp Afternoon section

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