Transcript Cxy_iGEM_13-6

miscellaneous…
& iGEM Design V1.0
- Xiuye
13.6.2008
Rough schedule
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Discussion today (13/6)
Discussion tmr (14/6)
Discussion the day after tmr (15/6)
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Detailed work assignment about parts etc.
1st Draft by next Friday (23/6)
2nd Draft by next next Monday (27/6)
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Submission by that Friday (30/6)
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Original project proposal
Part 1: random#-generator
 Options of Part 2 (read-out)
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 Simple
read-out (positive feedback?)
 Combination of spatial independent #s
 Combination of temporal independent #s
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All monitored by fluorescence
Part 1 Design ideas
By (manually?) capturing phases in
oscillation…
 Fluctuations in Random Diffusion etc…
 Identical promoters…mutually exclusive?
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Collins’ Toggle-switch
random review of ~~~Noise~~~
“People are fascinated by how we do what we
do despite this noise.”
— James Collins
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Burst of literature in the recent years…
Foundations for engineering biology
 Drew
Endy, Nature 2005
May 27, 2008
The 2008 HHMI Investigators
James J. Collins, Ph.D.
Boston University
Boston, MA
James Collins combines expertise in engineering, physics, and biology to
design and build synthetic gene networks for applications in biotechnology
and medicine and to reverse engineer the endogenous gene networks in
bacteria that regulate their responses to antibiotics. More
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Michael B. Elowitz, Ph.D.
California Institute of Technology
Pasadena, CA
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http://www.hhmi.org/news/elowitz_bio.html
Extrinsic/intrinsic noise
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Elowitz et al, (2005) Science
“working” noise
e.g. transformation
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He's particularly interested in learning
how cells make decisions about what
type of cell to become. To delve into
this phenomenon, he chose a model
bacterium called Bacillus subtilis,
which sometimes switches on a
program that lets it gobble up DNA
from its environment. This state, called
competence, happens seemingly
spontaneously. And in a dish of
genetically identical B. subtilis, only 5–
10 percent of the bacteria will flip into
competence mode. "Why is it when
you put these identical cells in the
same environment, they do different
things?" asks Elowitz. "It illustrates a
basic phenomenon in biology. There's
a lot of variability among cells that is
not genetic."
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…(they) discovered that competence
is triggered by natural and random
fluctuations inside individual cells. That
is, sometimes one of the bacteria
randomly makes a larger-than-normal
amount of a specific protein. This
excess protein then triggers the
genetic competence program, causing
the cell to become competent for a
while and then switch back to its
original state.
….. They showed that key properties
of the cell, such as how actively it
turns out different proteins, are
intrinsically random. This principle
overturns decades of dogma that said
that genes—and networks of genes—
operate in a completely predictable
and fixed fashion.
http://www.elowitz.caltech.edu/publications/CompetenceExcitable.pdf
Phenotypic noise
We varied independently the
rates of transcription and translation of a single fluorescent
reporter gene in the chromosome of Bacillus subtilis, and we
quantitatively measured the resulting changes in the phenotypic
noise characteristics. We report that of these two parameters,
increased translational efficiency is the predominant
source of increased phenotypic noise. This effect is consistent
with a stochastic model of gene expression in which
proteins are produced in random and sharp bursts.
Regulation of noise in the expression of a single gene
Ertugrul M. Ozbudak1, Mukund Thattai1, Iren Kurtser2, Alan D. Grossman2 & Alexander van
Oudenaarden1
Nature Genetics (2002)
http://www.nature.com/ng/journal/v31/n1/pdf/ng869.pdf
Figure 3. The burst size effect.
“working” noise
e.g. in l-phage
In some circumstances, noise can be highly desirable: an organism could use
high translation rates and large concentration fluctuations
as a means of creating nongenetic individuality in a population19,20.
This is seen with the cI gene of λ-phage4,21: upon
infection of a host cell, the cI mRNA is transcribed with an efficient
RBS upstream of the initiation codon, thus creating a highnoise
state; however, the lysogenic phenotype, once established,
is maintained in a low-noise state (since transcription then
begins at the initiation codon itself, producing inefficiently
translated mRNA4).
More Background
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E.coli statistics
http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi
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About Diffusion in E.coli
Ratio of decay rates for different diffusion modes. Since the
diffusion time is proportional to L2, long cells make higher
decay modes accessible to measurement. To obtain the ratio of
the decay rates of the first and second Fourier modes on the
same cell, cells were treated with cephalexin, a drug which
inhibits septation and causes cells to grow into long filaments.
Eleven cells ranging in length from 7.5 to 11 mm were selected,
and laser pulses were applied alternately at the cell pole and
the cell center until GFP was completely photobleached. The
first and second Fourier modes were analyzed from recovery
data after photobleaching of the cell pole and center, respectively.
An example of this experiment is presented in Fig. 1E
and F. Values obtained for Da were 7.2 6 1.3 mm2/s (average 6
SD; n 5 8) for mode 1 and 6.8 6 1.2 mm2/s for mode 2,
consistent with the experiments done without cephelaxin on
cells roughly half as long.
http://www.elowitz.caltech.edu/publications/ColiDiff.pdf
Margolin, 2006 CurBio
Shih et al, PNAS 2003
Huitema et al, 2006 Cell