Winston Group Meeting 09.19.06
Overview <ul><li>2 projects SAGA swap </li></ul><ul><li>mtDNA rewrite </li></ul><ul><li>justification </li></ul><ul><li>de...
mtDNA rewrite: justification Dedicated system for synthesis <ul><li>Ideally want system channel/chassis channel to be </li...
mtDNA rewrite: justification “  (we often) imagine the mitochondrion as a lonely participant in the cell, working tireless...
mtDNA rewrite: justification http://db.yeastgenome.org/cgi-bin/gbrowse/yeast/?name=chrMito%3A1..85779 mt DNA 85,779 bps 8 ...
mtDNA rewrite: design Existing marker for mtDNA manipulation Steele et al PNAS (1996) 93:5253 2 other mtDNA markers: GFP, ...
mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><...
mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><...
mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1  Step 3: biolist...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain DSF160 from Tom Fox, Cornell MATalpha  ade2-101 leu2∆ ura3-52 arg8...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain DFS160 MH339 1 2 3 4 5 6 G418 + dALA G418 30° 2d Also checked by P...
mtDNA rewrite: design Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1  BBpre BBsuff ...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Transformation results with screen DFS160 DFS160 hem1::KanMX No DNA 0   6 (hole in plate) pRS415 0, 0 ...
mtDNA rewrite: data Transformation results with screen DFS160 DFS160 hem1::KanMX No DNA 0   6 (hole in plate) pRS415 0, 0 ...
mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1   Step 3: biolis...
mtDNA rewrite: data Check for mtDNA by mating ade2 leu2 ∆  hem1 ::KanMX kar1-1 pRS415  rho 0  +mHEM1? x Desired strain wil...
mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient hem1 -leu + dALA -leu  30° 3d +LEU2 +LEU2 +mHEM1 M...
mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient G418 + dALA G418 30° 3d hem1 +LEU2 +LEU2 +mHEM1 ma...
mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient YPEG + dALA YPEG  30° 3d hem1 +LEU2 +LEU2 +mHEM1 m...
mtDNA rewrite: data mHEM1 seems to complement nuclear  hem1∆ but unclear why integration into mtDNA makes cells more red w...
Thanks and see you next summer!
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  • Does COX3 flank have promoter??
  • Macintosh_HD-Users-nkuldell-Desktop-WinstonGM_2006_pt2.ppt

    1. 1. Winston Group Meeting 09.19.06
    2. 2. Overview <ul><li>2 projects SAGA swap </li></ul><ul><li>mtDNA rewrite </li></ul><ul><li>justification </li></ul><ul><li>design </li></ul><ul><li>data </li></ul><ul><li>next steps/next summer :( </li></ul>Cindy Kolodziejski Bittersweet Chocolate Drop, 2004 earthenware with metal support 23.5 x 5.0 x 9.5 in.
    3. 3. mtDNA rewrite: justification Dedicated system for synthesis <ul><li>Ideally want system channel/chassis channel to be </li></ul><ul><li>orthogonal…e.g. draw from different pools of reagents </li></ul><ul><li>decoupled…e.g. run system independent of growth rate </li></ul><ul><li>generic…e.g. run same system in different chassis </li></ul>
    4. 4. mtDNA rewrite: justification “ (we often) imagine the mitochondrion as a lonely participant in the cell, working tirelessly to produce the energy required for life.” McBride et al Curr Biol 2006 <ul><li>Ideally want system channel/chassis channel to be </li></ul><ul><li>orthogonal…e.g. draw from different pools of reagents </li></ul><ul><li>decoupled…e.g. run system independent of growth rate </li></ul><ul><li>generic…eventually run same system in different chassis </li></ul>+/- +/- X
    5. 5. mtDNA rewrite: justification http://db.yeastgenome.org/cgi-bin/gbrowse/yeast/?name=chrMito%3A1..85779 mt DNA 85,779 bps 8 verified protein encoding genes 24 tRNA genes 2 rRNA genes ~20 nucleic acid processing factors encoded by introns
    6. 6. mtDNA rewrite: design Existing marker for mtDNA manipulation Steele et al PNAS (1996) 93:5253 2 other mtDNA markers: GFP, BARSTAR
    7. 7. mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><li>LYS12 </li></ul><ul><li>PUT1 </li></ul><ul><li>HEM1 </li></ul>
    8. 8. mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><li>LYS12 </li></ul><ul><li>PUT1 </li></ul><ul><li>HEM1 </li></ul>5-aminolevulinate synthase
    9. 9. mtDNA rewrite: design New marker for mtDNA <ul><li>Considered </li></ul><ul><li>MEL1 </li></ul><ul><li>xFP </li></ul><ul><li>LYS12 </li></ul><ul><li>PUT1 </li></ul><ul><li>HEM1 </li></ul>5-aminolevulinate synthase 1647 bp, 549 aa localized to mitochondrial matrix
    10. 10. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation
    11. 11. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain DSF160 from Tom Fox, Cornell MATalpha ade2-101 leu2∆ ura3-52 arg8∆ ::URA3 kar1-1 [rho0] outgrow in YPD ON select on G418+dALA hem1::KanMX
    12. 12. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain DFS160 MH339 1 2 3 4 5 6 G418 + dALA G418 30° 2d Also checked by PCR with KanMX/dwstm primer And will check by Western… hem1::KanMX
    13. 13. mtDNA rewrite: design Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 BBpre BBsuff mHEM1 HA COX3
    14. 14. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Select for nuclear txn (Leu + ), screen for mt txn (dALA indep) N mt leu2 ∆ hem1 ::KanMX rho 0 Tungsten powder mHEM1 LEU2
    15. 15. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Take 1 Visitor with Yasunori Hayashi and Ken Okamoto
    16. 16. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Take 2 Visitor with Marc Vidal and Stu Milstein
    17. 17. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Take 2 Visitor with Marc Vidal and Stu Milstein
    18. 18. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Take 2 Visitor with Marc Vidal and Stu Milstein
    19. 19. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Take 2 Visitor with Marc Vidal and Stu Milstein
    20. 20. mtDNA rewrite: data Transformation results with screen DFS160 DFS160 hem1::KanMX No DNA 0 6 (hole in plate) pRS415 0, 0 6, 6 pRS415, BBa_Y00100 nd 3, 4 no screen No DNA 25 (shot last) 0 pRS415 28, 90 58, 40 pRS415, BBa_Y00100 nd 78, 35
    21. 21. mtDNA rewrite: data Transformation results with screen DFS160 DFS160 hem1::KanMX No DNA 0 6 (hole in plate) pRS415 0, 0 6, 6 pRS415, BBa_Y00100 nd 3, 4 -leu+dALA + 1 2 3 4 5 6 7
    22. 22. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation Select for nuclear txn (Leu+), screen for mt txn (dALA indep) N mt leu2 ∆ hem1 ::KanMX rho 0 Tungsten powder mHEM1 LEU2
    23. 23. mtDNA rewrite: data Check for mtDNA by mating ade2 leu2 ∆ hem1 ::KanMX kar1-1 pRS415 rho 0 +mHEM1? x Desired strain will be: red/Leu + /G418 R /dALA indep/respiration deficient 2 of 7 show these p-types N mt N mt kar1-1 leu1 rho + (intronless)
    24. 24. mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient hem1 -leu + dALA -leu 30° 3d +LEU2 +LEU2 +mHEM1 Mated cand 1 Mated cand 2
    25. 25. mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient G418 + dALA G418 30° 3d hem1 +LEU2 +LEU2 +mHEM1 mated cand 1 mated cand 2
    26. 26. mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient YPEG + dALA YPEG 30° 3d hem1 +LEU2 +LEU2 +mHEM1 mated cand 1 mated cand 2
    27. 27. mtDNA rewrite: data mHEM1 seems to complement nuclear hem1∆ but unclear why integration into mtDNA makes cells more red why respiration requires dALA <ul><li>Follow up with </li></ul><ul><li>Western…. protocol for isolating of mt proteins? </li></ul><ul><li>PCR of mtDNA… protocol for isolation mtDNA?) </li></ul><ul><li>Microarray </li></ul><ul><li>Other targets in mtDNA </li></ul>
    28. 28. Thanks and see you next summer!

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