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!