Winston Group Meeting 09.19.06. Overview 2 projectsSAGA swap mtDNA rewrite justification design data next steps/next summer :( Cindy Kolodziejski Bittersweet.

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Presentation transcript:

Winston Group Meeting

Overview 2 projectsSAGA 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 Dedicated system for synthesis Ideally want system channel/chassis channel to be 1. orthogonal…e.g. draw from different pools of reagents 2. decoupled…e.g. run system independent of growth rate 3. generic…e.g. run same system in different chassis

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 Ideally want system channel/chassis channel to be 1. orthogonal…e.g. draw from different pools of reagents 2. decoupled…e.g. run system independent of growth rate 3. generic…eventually run same system in different chassis +/- X

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

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

mtDNA rewrite: design New marker for mtDNA Considered > MEL1 > xFP > LYS12 > PUT1 > HEM1

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

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

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

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] hem1::KanMX outgrow in YPD ON select on G418+dALA

mtDNA rewrite: data Step 1: hem1::KanMX deletion strain hem1::KanMX DFS160 MH G418 + dALAG418 30° 2d Also checked by PCR with KanMX/dwstm primer And will check by Western…

mtDNA rewrite: design Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 mHEM1HA BBpreBBsuff COX3

mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation N mt leu2∆ hem1::KanMX rho 0 Tungsten powder mHEM1 LEU2 Select for nuclear txn (Leu + ), screen for mt txn (dALA indep)

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

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

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

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

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

mtDNA rewrite: data Transformation results with screenDFS160DFS160 hem1::KanMX No DNA0 6 (hole in plate) pRS4150, 0 6, 6 pRS415, BBa_Y00100nd 3, 4 no screen No DNA25 (shot last) 0 pRS41528, 90 58, 40 pRS415, BBa_Y00100nd 78, 35

mtDNA rewrite: data Transformation results with screenDFS160DFS160 hem1::KanMX No DNA0 6 (hole in plate) pRS4150, 0 6, 6 pRS415, BBa_Y00100nd 3, 4 -leu+dALA

mtDNA rewrite: data Step 1: hem1::KanMX deletion strain Step 2: synthesis of mitochondrially encoded HEM1 Step 3: biolistic transformation N mt leu2∆ hem1::KanMX rho 0 Tungsten powder mHEM1 LEU2 Select for nuclear txn (Leu+), screen for mt txn (dALA indep)

mtDNA rewrite: data Check for mtDNA by mating N mt ade2 leu2∆ hem1::KanMX kar1-1 pRS415 rho 0 +mHEM1? N mt kar1-1 leu1 rho + (intronless) x Desired strain will be: red/Leu + /G418 R /dALA indep/respiration deficient 2 of 7 show these p-types

mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient hem1 -leu + dALA-leu 30° 3d +LEU2 +mHEM1 Mated cand 1 Mated cand 2

mtDNA rewrite: data red/Leu + /G418 R /dALA indep/respiration deficient G418 + dALAG418 30° 3d hem1 +LEU2 +mHEM1 mated cand 1 mated cand 2

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

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 for isolating of mt proteins? PCR of mtDNA…protocol for isolation mtDNA?) Microarray Other targets in mtDNA

Thanks and see you next summer!