Getting organizedfrom mitochodrial gene expression to public dialog

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Getting organized--from mitochodrial gene
expression to public dialog

• Winston Retreat
• 06.18.08

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"Out of clutter, find simplicity... From discord
find harmony... In the middle of difficulty lies
opportunity." ---Albert Einstein
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Many genes, many sources Pathway for the
production of the anti-malarial drug precursor,
amorphadiene
Red toxic intermediates
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One Approach Flux BalanceEnzymes have
different levels of activity (turnover fluxes)
Loss of intermediates to bulk solution Enzymes
freely diffuse in the cell Especially problematic
for cytotoxic intermediates
slide from John Dueber, UCB
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Synthetic ScaffoldsCo-localize pathway enzymes
and reduce intermediate runoff
slide from John Dueber, UCB
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Synthetic ScaffoldsCo-localize pathway enzymes
and reduce intermediate runoff
since scaffold is modular, can also mulitmerize
ENZYME 2
slide from John Dueber, UCB
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Complementary approach compartmentalization
Build spatially-isolated, biochemically-distinct
microenvironment
1. functionalized (poly guanylic acid)
2. coat (PMOXA-PDMS-PMOXA)
3. payload (fluorescent BSA)
functionalized polymer vesicle (fPV)
artificial organelle 200 nm diameter innocuous
in cell culture
Ben-Haim et. al. Nano Lett (2008) 8(5) 1368
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Complementary approach compartmentalization
Build spatially-isolated, biochemically-distinct
microenvironment
functionalized polymer vesicle (fPV)
Ben-Haim et. al. Nano Lett (2008) 8(5) 1368
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Complementary approach compartmentalization
Build spatially-isolated, biochemically-distinct
microenvironment
macrophage engulf fPV with fluor-BSA, stable
48hrs
Ben-Haim et. al. Nano Lett (2008) 8(5) 1368
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Alternative approach to compartmentalization
Use existing microenvironment
self-assembling, stable, spatially isolated,
biochemically-distinct
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Alternative approach to compartmentalization
Use existing microenvironment
self-assembling, stable, spatially isolated,
biochemically-distinct, genetically manipulated
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S. cerevisiae mt current contents

• mt genome includes
• 8 protein coding genes
• 7 oxphos, 1 riboprot
• 2 rRNAs
• 24 tRNAs

• Can read and write mt DNA,
• but what to say??
• Selectable marker
• Tunable expression

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S. cerevisiae mt selectable marker
Existing markers for mtDNA manipulation
2 other mtDNA markers GFP, BARSTAR
Steele et al PNAS (1996) 935253
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S. cerevisiae mt selectable marker
HEM1 549 aa nuclear-encoded protein normally
localized to mt matrix
Step 1 hem1KanMX ?0 deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
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S. cerevisiae mt tunable expression
Existing mt gene expression

• transcriptional regulation _at_ initiation

PNAS (1990) 879338
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S. cerevisiae mt tunable expression
Existing mt gene expression

• transcriptional regulation _at_ initiation

• translational _at_ inner membrane

http//www.mbg.cornell.edu/faculty-staff/faculty/f
ox.cfm
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S. cerevisiae mt tunable expression
Part 3 dsRNase
Part 2 guide RNA
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S. cerevisiae mt tunable expression
Expression vector for mitochondrial Rnt1
signal sequence
HA tag
?NLS (11 aa)
RNT1
pRS416
?NLS in Henras et al RNA (2004) 101572
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S. cerevisiae mt short term goals
mtHEM1 marker
mtRNT1p
/- dox, check protein, mtRnt1 RNA, plate
phenotypes repeat microarray? localization to
mt?
then look at plate phenotypes, western, PCR of
mtDNA
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its just a theory
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• An ongoing communication network of
  science-interested parties
• Objectives
• Provide a shared appreciation of science
• Inform and engage the public in and about science
  
• Make science more accessible to everyone

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the end
"Out of clutter, find simplicity... From discord
find harmony... In the middle of difficulty lies
opportunity." ---Albert Einstein
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its just a theory
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its just a theory
In November 8, 2005 the Kansas Board of Education
approved the following changes to its science
standards 1. Add to the mission statement a
goal that science education should seek to
"inform. 2. Provide a definition of science
that does not preclude supernatural
explanations. 3. Allow intelligent design to
be presented as an alternative explanation to
evolution as presented in mainstream biology
textbooks, without endorsing it. 4. State
that evolution is a theory and not a fact. 5.
Require informing students of purported
scientific controversies regarding evolution.
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its just a theory
PLoS Biol (2008) 6(5) e124
Winning in the courts Losing in the classroom
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SW Lee Allison took up the post of Kansas state
geologist just two weeks before the school
board's vote. He thinks scientists mistakenly
think good science will stand on its own merit
they shy away from political debate, and they're
no match for what he sees as the well-oiled
political machine of the creation lobby. Lee
Allison 'Kansas State Geologist' Lee Allison
Over the past twenty or thirty years, we've seen
a very sophisticated, well funded, well trained,
actually well educated group of people who are
trying to push forward a religious agenda.
They've been building up their support, by
putting people into elected offices for many
years, for a decade, of getting enough people in
until they have a majority and when none of us
expected this to happen in Kansas, it caught us
all by surprise. SW There seems to be uh,
almost a sense of victory if these people can
persuade scientists to admit that 'evolution is
just a theory' is that a misuse of the word
theory? LA The anti-evolution group is misusing
the concept of a theory. They're using 'theory'
as is generally used out on the streets by
anybody 'Gee, I have a theory that, uh its going
to rain tonight.' Well, it's it's just a hunch,
it's a speculation, a guess--- that's not how we
use 'theory' in science. A theory is a well
documented explanation of natural phenomena based
on multiple observations, multiple tests,
developed from many arenas and brought together
to develop a theory.
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principal energy policy and planning arm of
state government. The council's task is to
"formulate and coordinate a comprehensive state
energy plan."
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• Today's Topics
• 1. Re systemic limitation of biobricks
  for combinatorial logic?
• (Dr. Markus Schmidt)
• 2. Re systemic limitation of biobricks
  for combinatorial logic?
• (Deepak Chandran)
• --------------------------------------------------
  --------------------
• Message 1
• Date Tue, 20 May 2008 134615 0200
• From "Dr. Markus Schmidt" ltmarkus.schmidt_at_idialog
  .eugt
• Subject Re BBF Standards systemic limitation
  of biobricks for
• combinatorial logic?
• To standards_at_biobricks.org
• Thanks JC for bringing the issues back into the
  disussion. The system-
• wide visibility of all gate outputs is a serious
  obstacle to the
• development of the standardized biopart concept.
  When I posted the
• message on lacking specifity in February there
  was hardly any
• reaction, which really surprised me. Sure as long
  as the experimental
• phase of biobricks now runs systems that contain
  only few parts, all