Winston Group Meeting
09.19.06
Overview
2 projects SAGA swap
mtDNA rewrite• justification• design• data• next steps/next summer :(
Cindy KolodziejskiBittersweet Chocolate Drop, 2004
earthenware with metal support23.5 x 5.0 x 9.5 in.
mtDNA rewrite: justificationDedicated 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
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
mtDNA rewrite: designExisting marker for mtDNA manipulation
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Steele et al PNAS (1996) 93:5253
2 other mtDNA markers: GFP, BARSTAR
mtDNA rewrite: designNew marker for mtDNA
Considered
> MEL1
> xFP
> LYS12
> PUT1
> HEM1
mtDNA rewrite: designNew marker for mtDNA
Considered
> MEL1
> xFP
> LYS12
> PUT1
> HEM1 5-aminolevulinate synthase
mtDNA rewrite: designNew marker for mtDNA
Considered
> MEL1
> xFP
> LYS12
> PUT1
> HEM1 5-aminolevulinate synthase
1647 bp, 549 aa
localized to mitochondrial matrix
mtDNA rewrite: dataStep 1: hem1::KanMX deletion strain
Step 2: synthesis of mitochondrially encoded HEM1
Step 3: biolistic transformation
mtDNA rewrite: dataStep 1: hem1::KanMX deletion strain
DSF160 from Tom Fox, CornellMATalpha ade2-101 leu2∆ ura3-52 arg8∆ ::URA3 kar1-1 [rho0]
hem1::KanMX
outgrow in YPD ON
select on G418+dALA
mtDNA rewrite: dataStep 1: hem1::KanMX deletion strain
hem1::KanMX
DFS160
MH339
1 23
456
G418 + dALA G418 30° 2d
Also checked by PCR with KanMX/dwstm primer
And will check by Western…
mtDNA rewrite: designStep 1: hem1::KanMX deletion strain
Step 2: synthesis of mitochondrially encoded HEM1
mHEM1 HABBpre BBsuff
COX3
mtDNA rewrite: dataStep 1: hem1::KanMX deletion strain
Step 2: synthesis of mitochondrially encoded HEM1
Step 3: biolistic transformation
Nmt
leu2∆ hem1::KanMX rho0
Tungsten powder
mHEM1
LEU2
Select for nuclear txn (Leu+), screen for mt txn (dALA indep)
mtDNA rewrite: dataStep 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: dataStep 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
QuickTime™ and aMotion JPEG OpenDML decompressor
are needed to see this picture.
mtDNA rewrite: dataStep 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
QuickTime™ and aMotion JPEG OpenDML decompressor
are needed to see this picture.
mtDNA rewrite: dataStep 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
QuickTime™ and aMotion JPEG OpenDML decompressor
are needed to see this picture.
mtDNA rewrite: dataStep 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: dataTransformation 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
mtDNA rewrite: dataTransformation 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
+
12
3
45 6
7
mtDNA rewrite: dataStep 1: hem1::KanMX deletion strain
Step 2: synthesis of mitochondrially encoded HEM1
Step 3: biolistic transformation
Nmt
leu2∆ hem1::KanMX rho0
Tungsten powder
mHEM1
LEU2
Select for nuclear txn (Leu+), screen for mt txn (dALA indep)
mtDNA rewrite: dataCheck for mtDNA by mating
Nmt
ade2 leu2∆ hem1::KanMX kar1-1 pRS415
rho0 +mHEM1?
Nmt
kar1-1 leu1
rho+ (intronless)
x
Desired strain will be:
red/Leu+/G418R/dALA indep/respiration deficient
2 of 7 show these p-types
mtDNA rewrite: data
red/Leu+/G418R/dALA indep/respiration deficient
hem1
-leu + dALA -leu 30° 3d
+LEU2+LEU2
+mHEM1
Mated
cand 1
Mated
cand 2
mtDNA rewrite: data
red/Leu+/G418R/dALA indep/respiration deficient
G418 + dALA G418 30° 3d
hem1
+LEU2+LEU2
+mHEM1
mated
cand 1
mated
cand 2
mtDNA rewrite: data
red/Leu+/G418R/dALA indep/respiration deficient
YPEG + dALA YPEG 30° 3d
hem1
+LEU2+LEU2
+mHEM1
mated
cand 1
mated
cand 2
mtDNA rewrite: datamHEM1 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!