1 / 46

Erwin Schrodinger

What is Life?. Erwin Schrodinger. 1944. Nobel Prize in Physics 1933 Atomic Theory. Max Delbruck Nobel Prize in Medicine 1969. Seymour Benzer. rII……rapid lysis…. makes large plaques. E.coli. T4. K B. + -. WT. - +.

razi
Télécharger la présentation

Erwin Schrodinger

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. What is Life? Erwin Schrodinger 1944 Nobel Prize in Physics 1933 Atomic Theory

  2. Max Delbruck Nobel Prize in Medicine 1969

  3. Seymour Benzer rII……rapid lysis…. makes large plaques E.coli T4 K B + - WT - + rII

  4. Plate 0.1 ml and count # plaques 103 109 106 Example-plate has 50 plaques, therefore 500 phage per ml at 109 or total = 5x 1011 per ml

  5. E.coli Back to Benzer T4 K B + - WT - + rII Plate concentrated wild type on strain B to select rII mutants Grow rII mutants to high density (strain B) Plate onto strain K to select rare revertants Some rII alleles revert (low frequency) others never revert Two different non-revertable alleles and do mixed infection on Strain K Get very rare plaques that result from recombination

  6. rII Does not grow on K rII-1 rII-2 Does not grow on K Does not grow on K Grows on K Internal deletion

  7. In the early days of phage genetics….. • Two types of mutants… • Altered plaque size and shape • Host range…..grow on certain strains of E. coli • Ultimately the goal became to identify every gene in the genome • Filling in the map with conditional mutants • Temperature sensitive mutants • Nonsense mutants

  8. From the very beginning of Molecular Biology and Genetics The goal has been to have a complete understanding of the genome This means assigning a function to every gene in the genome genotype phenotype DNA Function

  9. Assigning functions to a gene When is it expressed? Where is it expressed? Is the protein modified? Protein-Protein Interactions? Phenotype when protein is reduced? Phenotype when the protein is overexpressed?

  10. GatewayTM Technology Recombineering in vitro A diversion to phage lambda Life style choice… Lysis vs lysogeny Lysis-plaques Lysogeny-phage infects the cell but is dormant …The cell survives until there is some stress (uv light) …Lysis Lysogens (bacteria with dormant phage) are phage resistant

  11. Lysogeny-Lysis Lysis attP attB Int attR attL Lysogen Stress Xis Lysis

  12. Step 1…generate an “entry clone” with YFG

  13. Step 2 recombine YFG into a destination vector

  14. Destination vectors for every use

  15. Assigning functions to a gene When is it expressed? Microarray experiments Where is it expressed? Epitope tagged protein Is the protein modified? Gel shifts and mass spectrometry Protein-Protein Interactions? GST or other affinity purifications Phenotype when protein is reduced? siRNA Phenotype when the protein is overexpressed? Strong promoter But problems remain for tissue culture cells

  16. Transfection (not transformation) Stable (hard) or transient (easy)? Transient: Fraction of transfected cells is variable Expression levels differs in individual cells What cell types do you choose? What is the isogenic wild type control?

  17. HeLa cells karyotype from ATCC Modal number of chromosomes= 82 Range = 70 to 164. 100% aneuploidy in 1385 cells examined.

  18. Reducing expression by shRNA Libraries

  19. URA3 URA3 Recall yeast one-step gene replacements YFG1

  20. in vitro approach Use TAP purifications to make protein chips GST tagged protein kinases

  21. Integrating Kinase Expression Array and TAP data

More Related