Getting organized--from mitochodrial gene expression to public dialog Winston Retreat 06.18.08.

Getting organized--from mitochodrial gene expression

to public dialogWinston 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

from

Artemisia annua

black from

(overexp’d)

Red = toxic intermediates

One Approach: Flux Balance

Enzymes have different levels of activity (turnover fluxes)

Loss of intermediates to bulk solutionEnzymes freely diffuse in the cellEspecially problematic for cytotoxic intermediates

slide from John Dueber, UCB

Synthetic ScaffoldsCo-localize pathway enzymes and reduce

intermediate runoff


Synthetic ScaffoldsCo-localize pathway enzymes and reduce

intermediate runoff


since scaffold is modular, can also mulitmerize ENZYME 2

Complementary approach: compartmentalization

Build spatially-isolated, biochemically-distinct microenvironment

Ben-Haim et. al. Nano Lett (2008) 8(5): 1368

functionalized polymer vesicle (fPV) “artificial organelle”

~200 nm diameter

innocuous in cell culture

3. payload (fluorescent BSA)

2. coat (PMOXA-PDMS-PMOXA)1. functionalized (poly guanylic acid)




functionalized polymer vesicle (fPV)




macrophage engulf fPV with fluor-BSA, stable ~48hrs

Alternative approach to compartmentalization

Use existing microenvironment

self-assembling, stable, spatially isolated, biochemically-distinct

Alternative approach to compartmentalization

Use existing microenvironment

self-assembling, stable, spatially isolated, biochemically-distinct, genetically manipulated

Budweiser beer

factory, Czech

Republic

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 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

S. cerevisiae mt: selectable markerHEM1

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

15X

strong if +2= purine

Existing mt gene expression

• transcriptional regulation @ initiation

PNAS (1990) 87:9338

mt RNAP RPO41 = catalytic subunit MTF1 = specificity factor

mt promoters TAtAaGtN (+1)

nuclear-encoded

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


Part 3: dsRNase

Part 1: mRNA target

e.g. mtGFP

Part 2: guide RNA

Expression vector for mitochondrial Rnt1

pRS416

RNT1

signal sequence + HA tag

∆NLS (11 aa)

∆NLS in Henras et al RNA (2004) 10:1572


S. cerevisiae mt: short term goals

mtHEM1 marker mtRNT1p

then look at plate phenotypes, western, PCR of mtDNA

+/- dox, check

protein,

mtRnt1 RNA,

plate phenotypes

repeat microarray?

localization to mt?

“it’s just a theory”

• 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

the end

"Out of clutter, find simplicity...From discord find harmony...In the middle of difficulty lies opportunity."

---Albert Einstein


“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.

Winning in the courts Losing in the classroom


PLoS Biol (2008) 6(5): e124

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."

• 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]

• 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 • 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.

• If we take electronic integrated circuits as an example (and not as a • metapher), than producing compartiments is the goal.

• Which ways are there to produce compartiments?

• To begin with I would say there are spacial, chemical, sematic or time- • based compartiments.

• 1. Spacial:• 1.1. new organelles. This is a nice idea but how many organelles can • you engineer into a cell? Tens, hundreds, but certainly not millions.• 1.2. cell-cell communications. of course this is an option but it is • basically the same situation as in the organelles, although with the • option to increase the number of differnet cells without the packing • problems of organelles. Basically the way by wich the cells • communicate is the bottleneck.• 2. Chemical:• 2.1. Number of molecules to be used as an information carrier is • extremely large but at the cost of reduced specifity and increasing • cross talk.• 2.2. Quorum sensing. This is done by molecules and only because it • involves the extracellular environment it doesn't mean it solves the • problem of open logic gates.• 3. Semantic:• 3.1. I guess the zinc finger story comes in here, an approach that • target the genetic code. The information you can store on a lets say • x bp long DNA is 4^x (may be reduced for some mutation-robustness • meassures) and could provide enough specifity to deal with ultra large • scale circuits. This is actually a promising approach, programming an • RNA computer• 4. Time-based• 4.1. PoPS, for Polymerase Per Second. This is a nice idea (and the • comic is fun) and transcends/converts the problem of chemical • specifity to a unambigous signal unit. OK, lets say you found a way to • meassure the PoPS rigt on the DNA. Say you meassuered x PoPS. Then you • have a subsequent PoPS analyzer that makes e.g. the following • decision: if x<y output=0; if y<x<z output undefined; if z<x output=1. • Problem solved, isn't it? Well it is but only in the case if the PoPS • analyzer (counter) sits right after the PoPS relevant piece of DNA, • otherwise you would have to transfer the result x into a chemical • signal to transport it to another part of DNA or elsewhere and then • you run into the same problem of open logic gates. So if you avoid • that and realize a linear logic line (a Ford like assembly line) you • are quite limited in running your software.

• However, what I think can be done is to combine all these approaches • in order to push the limit of the maximum number (Nmax) of realizable • "logic gates" or operations a little bit. Maybe each approach can help • us to push the Nmax by a factor of 100 or 1000 (or maybe more).• However, this is way a different story than with Moore's law, where • basically the reduction of size ( and packaging) of logic gates was • and is the main driving force to improve the number of transistor per • chip.

• Cheers, Markus

Getting organized--from mitochodrial gene expression to public dialog Winston Retreat 06.18.08.

Documents

cerevisiae mt

mt matrixstep

mt dna

hrsalternative approach

distinctalternative

john dueber

mtdna manipulationsteele

henras et