synthetic systems for teaching and learning
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Synthetic Systems for Teaching and Learning. Winston Retreat June 25th, 2007 Natalie Kuldell. Unreal Irrational. Undergraduate teaching with SAGA deletions. Regulating RNA degradation in yeast mitochondria. Why hack the yeast mitochondria?. - PowerPoint PPT PresentationTRANSCRIPT
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Synthetic Systems for Teaching and Learning
Winston Retreat
June 25th, 2007
Natalie Kuldell
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Regulating RNA degradation in yeast
mitochondria
Undergraduate teaching with SAGA
deletions
Unreal Irrational
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Why hack the yeast mitochondria?
“ (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
http://grocs.dmc.dc.umich.edu/gallery/organelle/Interface2
Other mt functions
• coordinates with nuclear gene expression (disease/aging)
• spatially isolated enzymatic reaction center
• viability on nonfermentable carbon sources
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? =
Hacking yeast mitochondria
Wish list (incomplete)
1. Orthogonal draw from different pools of reagents
2. Decoupled run system independent of growth rate
3. Generic run same system in different chassis
4. Tunable vary operation at will
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Current contents: in mt from mt
mt genome includes
• 8 protein coding genes
7 oxphos, 1 riboprot
• 2 rRNAs
• 24 tRNAs
mt promoters TATAAGTA (+1)
mt RNAP RPO41 = catalytic subunit MTF1 = specificity factor
nuclear-encoded
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Targeted mtRNA degradation
Part 3: dsRNase
Part 1: mRNA target
e.g. mtGFP
Part 2: guide RNA
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Catala et al, MCB (2004) 15:3015
Snapshot of wild type role for Rnt1
• Localized to the nucleus even when overexpressed
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Snapshot of wild type role for Rnt1
• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)
• Localized to the nucleus even when overexpressed
Henras et al.RNA (2004) 10: 1572
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Snapshot of wild type role for Rnt1
• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)
• Localized to the nucleus even when overexpressed
• Processes some coding RNA, e.g. Mig2
Ge et al, Current Biology (2005) 15:140
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Catala et al, MCB (2004) 15:3015
Snapshot of wild type role for Rnt1
• Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs)
• Localized to the nucleus even when overexpressed
• Processes some coding RNA, e.g. Mig2
• Needed for normal cell cycle progression
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tTA
CMV
2x tetO
CYC1
pRS41n
modified RNT1
Expression vector for mitochondrial Rnt1
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Expression vector for mitochondrial Rnt1
pRS41n
RNT1
signal sequence + epitope tag
∆NLS (11 aa)
∆NLS in Henras et al RNA (2004) 10:1572
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Initial experiments with mtRnt1
1. Expression? by Western with epitope Ab
2. Phenotypes? Respiration, growth, existing markers
3. Overall? Microarray wt vs mtRnt
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Targeted mtRNA degradation
Part 3: dsRNase
Part 1: mRNA target
e.g. mtGFP
Part 2: guide RNA
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Protein import into mitochondria
Pfanner and Geissler Nat Rev
(2001) 2:339
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RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”
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RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”
~all mt tRNAs encoded on
nuclear genome
RNA receptor (“RIC”) in mt membrane Entelis et al Gene Engineering:
Principles and Methods (2001) 24:191
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RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”
no mt tRNAs encoded by mt
RIC + ytRNA--> repair mt defect in
human cell line
Mahata et al Science (2006) 314:471
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RNA import into mitochondria“poorly understood”/”mechanisms appear to differ”
all but one tRNA encoded on mt
genome
import depends on protein import
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Specialized import into mitochondria
protein:RNA conjugate
Piggyback on tRNA import
Bind to mtRNA binding protein
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Regulating RNA degradation in yeast
mitochondria
Undergraduate teaching with SAGA
deletions
Unreal Irrational
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Expression Engineering Experiment
Day 1 Day 2 Day 3
Day 4Day 5Day 6
RT
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Subunit Deleted? ADA3 1/1
GCN5 1/1
SPT3 (3 groups) 3/3
SPT8 (3 groups) 0/3
UBP8 (2 groups) 2/2
SUS1 (2 groups) 2/2
FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128
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Subunit Deleted? ADA3 1/1
GCN5 1/1
SPT3 (3 groups) 3/3
SPT8 (3 groups) 0/3
UBP8 (2 groups) 2/2
SUS1 (2 groups) 2/2
FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128
NY389
ura3-52 his4-917leu3∆1 trp1-63 spt8∆320::LEU2
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Day 3
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wt/sus1∆
Andrew Ji and Kate Broadbent, W/F Team Blue, 20.109 Spring ‘07
wt/sgf73∆
teacher
Follow-up with microarray
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Follow-up with spot tests
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Hi Natalie,I've attached my rewrite. Thanks! See you tomorrow,AndrewP.S. This was one of the most time-consuming assignments I've ever had to do, yet it was easily the most fun and rewarding thing I've ever accomplished for any school-related project.
Follow-up with spot tests
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Andrew Ji and Kate Broadbent
May 10, 2007
Sus1’s role in SAGA-dependent gene motility, transcription, and
expression under different cellular conditions
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From: Neal Lerner <[email protected]>Subject: Re: 109 writing assignmentDate: Thu, 11 Jan 2007 11:07:34 -0500To: natalie kuldell <[email protected]>
Natalie, as I prepare to give a writing-across-the-curriculum talk next week, I came across this quote from John Bean: WAC is about creating opportunities for students to have an "authentic desire to converse with interested readers about real ideas." Now, in most school settings that's pretty darn hard to achieve, but I think when students have the chance to write/talk about lab work and ideas they find interesting (as in 20.109), we have a shot at it.
See you on the 22nd.
Neal
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the end
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Current contents: in mt from nucleus
nuclear genome sends
• ~750 proteins to mt
87 of these are putative proteins of no known function