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Winston Group Meeting

Winston Group Meeting. 09.19.06. Overview. 2 projects SAGA swap mtDNA rewrite justification design data next steps/next summer :(. Cindy Kolodziejski Bittersweet Chocolate Drop, 2004 earthenware with metal support 23.5 x 5.0 x 9.5 in. mtDNA rewrite: justification.

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Winston Group Meeting

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  1. Winston Group Meeting 09.19.06

  2. Overview 2 projects SAGA swap mtDNA rewrite • justification • design • data • next steps/next summer :( Cindy Kolodziejski Bittersweet Chocolate Drop, 2004 earthenware with metal support 23.5 x 5.0 x 9.5 in.

  3. mtDNA rewrite: justification Dedicated system for synthesis Ideally want system channel/chassis channel to be orthogonal…e.g. draw from different pools of reagents decoupled…e.g. run system independent of growth rate generic…e.g. run same system in different chassis

  4. Ideally want system channel/chassis channel to be orthogonal…e.g. draw from different pools of reagents decoupled…e.g. run system independent of growth rate generic…eventually run same system in different chassis +/- +/- X 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

  5. mtDNA rewrite: justification mt DNA 85,779 bps 8 verified protein encoding genes 24 tRNA genes 2 rRNA genes ~20 nucleic acid processing factors encoded by introns http://db.yeastgenome.org/cgi-bin/gbrowse/yeast/?name=chrMito%3A1..85779

  6. mtDNA rewrite: design Existing marker for mtDNA manipulation 2 other mtDNA markers: GFP, BARSTAR Steele et al PNAS (1996) 93:5253

  7. mtDNA rewrite: design New marker for mtDNA • Considered • MEL1 • xFP • LYS12 • PUT1 • HEM1

  8. mtDNA rewrite: design New marker for mtDNA • Considered • MEL1 • xFP • LYS12 • PUT1 • HEM1 5-aminolevulinate synthase

  9. mtDNA rewrite: design New marker for mtDNA • Considered • MEL1 • xFP • LYS12 • PUT1 • HEM1 5-aminolevulinate synthase 1647 bp, 549 aa localized to mitochondrial matrix

  10. mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation

  11. hem1::KanMX 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

  12. hem1::KanMX mtDNA rewrite: data Step 1: hem1::KanMX deletion strain 6 5 MH339 4 DFS160 3 1 2 G418 + dALA G418 30° 2d Also checked by PCR with KanMX/dwstm primer And will check by Western…

  13. mHEM1 HA COX3 mtDNA rewrite: design Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 BBpre BBsuff

  14. mHEM1 mt Tungsten powder N leu2∆ hem1::KanMX rho0 LEU2 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)

  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. 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. 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. 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. 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. mtDNA rewrite: data Transformation results with screenDFS160 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. 2 1 3 + 4 7 5 6 mtDNA rewrite: data Transformation results with screenDFS160 DFS160 hem1::KanMX No DNA 0 6 (hole in plate) pRS415 0, 0 6, 6 pRS415, BBa_Y00100 nd 3, 4 -leu+dALA

  22. mHEM1 mt Tungsten powder N leu2∆ hem1::KanMX rho0 LEU2 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)

  23. mt mt kar1-1 leu1 rho+ (intronless) N N mtDNA rewrite: data Check for mtDNA by mating x ade2 leu2∆ hem1::KanMX kar1-1 pRS415 rho0 +mHEM1? Desired strain will be: red/Leu+/G418R/dALA indep/respiration deficient 2 of 7 show these p-types

  24. mtDNA rewrite: data +LEU2 +mHEM1 +LEU2 hem1 Mated cand 1 Mated cand 2 -leu + dALA -leu 30° 3d red/Leu+/G418R/dALA indep/respiration deficient

  25. +LEU2 +mHEM1 +LEU2 hem1 mated cand 1 mated cand 2 mtDNA rewrite: data G418 + dALA G418 30° 3d red/Leu+/G418R/dALA indep/respiration deficient

  26. +LEU2 +mHEM1 +LEU2 hem1 mated cand 1 mated cand 2 mtDNA rewrite: data YPEG + dALA YPEG 30° 3d red/Leu+/G418R/dALA indep/respiration deficient

  27. mtDNA rewrite: data mHEM1 seems to complement nuclear hem1∆ but unclear why integration into mtDNA makes cells more red why respiration requires dALA • Follow up with • Western….protocol forisolating of mt proteins? • PCR of mtDNA…protocol for isolation mtDNA?) • Microarray • Other targets in mtDNA

  28. Thanks and see you next summer!

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