Transcript ppt - OpenWetWare

Getting organized--from
mitochodrial gene expression
to public dialog
Winston Retreat
06.18.08
"Out of clutter, find simplicity...
From discord find harmony...
In the middle of difficulty lies opportunity."
---Albert Einstein
Many genes, many sources
Pathway for the production of the anti-malarial drug precursor,
amorphadiene
grey from
black from
(overexp’d)
from
Red = toxic intermediates
Artemisia annua
One Approach: Flux Balance
Enzymes 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
Synthetic Scaffolds
Co-localize pathway enzymes and reduce
intermediate runoff
slide from John Dueber, UCB
Synthetic Scaffolds
Co-localize pathway enzymes and reduce
intermediate runoff
since scaffold is modular, can also mulitmerize ENZYME 2
slide from John Dueber, UCB
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
Complementary approach:
compartmentalization
Build spatially-isolated, biochemically-distinct microenvironment
functionalized
polymer vesicle (fPV)
Ben-Haim et. al. Nano Lett (2008) 8(5): 1368
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
Alternative approach to
compartmentalization
Use existing microenvironment
self-assembling, stable, spatially isolated,
biochemically-distinct
Alternative approach to
compartmentalization
Use existing microenvironment
Budweiser
beer
factory,
Czech
Republic
self-assembling, stable, spatially isolated,
biochemically-distinct, genetically manipulated
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??
1. Selectable marker
2. Tunable expression
S. cerevisiae mt: selectable marker
Existing markers for mtDNA manipulation
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
2 other mtDNA markers: GFP, BARSTAR
Steele et al PNAS (1996) 93:5253
S. cerevisiae mt: selectable marker
HEM1
549 aa nuclear-encoded protein normally localized to mt matrix
Step 1: hem1::KanMX 0 deletion strain
Step 2: synthesis of mitochondrially encoded HEM1
Step 3: biolistic transformation
S. cerevisiae mt: tunable expression
Existing mt gene expression
• transcriptional regulation @ initiation
mt RNAP
RPO41 = catalytic subunit
MTF1 = specificity factor
mt promoters
nuclearencoded
TAtAaGtN
(+1)
15X
strong if +2= purine
PNAS (1990) 87:9338
S. cerevisiae mt: tunable expression
Existing mt gene expression
• transcriptional regulation @ initiation
• translational @ inner membrane
http://www.mbg.cornell.edu/faculty-staff/faculty/fox.cfm
S. cerevisiae mt: tunable expression
Part 1: mRNA target
e.g. mtGFP
Part 2: guide RNA
Part 3: dsRNase
S. cerevisiae mt: tunable expression
Expression vector for mitochondrial Rnt1
signal sequence
+ HA tag
RNT1
∆NLS
(11 aa)
pRS416
∆NLS in Henras et al RNA (2004) 10:1572
S. cerevisiae mt: short term goals
mtHEM1 marker
mtRNT1p
+/- dox, check
protein,
mtRnt1 RNA,
plate phenotypes
then look at plate phenotypes,
western, PCR of mtDNA
repeat microarray?
localization to mt?
“it’s just a theory”
• An ongoing communication network of scienceinterested parties
• Objectives:
– Provide a shared appreciation of science
– Inform and engage the public in and about science
– Make science more accessible to everyone
the end
"Out of clutter, find simplicity...
From discord find harmony...
In the middle of difficulty lies
opportunity."
---Albert Einstein
“it’s just a theory”
“Because Darwin’s Theory is a theory,
it continues to be tested as new
evidence is discovered. . . . Gaps in
the Theory exist for which there is no
evidence. . . . Intelligent design is an
explanation of the origin of life that
differs from Darwin’s view.”
--Read-aloud from Dover, PA school
board for 9th grade teachers
“it’s 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.
“it’s just a theory”
PLoS Biol (2008) 6(5): e124
Winning in the courts
Losing in the classroom
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.
…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 13:46:15 +0200
From: "Dr. Markus Schmidt" <[email protected]>
Subject: Re: [BBF Standards] systemic limitation of biobricks for
combinatorial logic?
To: [email protected]
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Thanks JC for bringing the issues back into the disussion. The systemwide 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
these problems do not arise, but thinking about the future and about
the prospect of this approach we should dedicate a susbstantial amount
of time and energy to come up with solutions.
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If we take electronic integrated circuits as an example (and not as a
metapher), than producing compartiments is the goal.
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Which ways are there to produce compartiments?
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To begin with I would say there are spacial, chemical, sematic or timebased compartiments.
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1. Spacial: