miscellaneous…& iGEM Design V1.0

- Xiuye13.6.2008

Rough schedule

Discussion today (13/6) Discussion tmr (14/6) Discussion the day after tmr (15/6) … Detailed work assignment about parts etc. 1st Draft by next Friday (23/6) 2nd Draft by next next Monday (27/6)

Submission by that Friday (30/6)

Original project proposal

Part 1: random#-generator Options of Part 2 (read-out)

Simple read-out (positive feedback?)Combination of spatial independent #sCombination of temporal independent #s

All monitored by fluorescence

Part 1 Design ideas

By (manually?) capturing phases in oscillation…

Fluctuations in Random Diffusion etc… Identical promoters…mutually exclusive?

Collins’ Toggle-switch

random review of ~~~Noise~~~

“People are fascinated by how we do what we do despite this noise.”

— James Collins

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 desig

n 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

Michael B. Elowitz, Ph.D.California Institute of TechnologyPasadena, CA

http://www.hhmi.org/news/elowitz_bio.html

Extrinsic/intrinsic noise Elowitz et al, (2005) Science

“working” noise e.g. transformation 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."

…(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 therates of transcription and translation of a single fluorescentreporter gene in the chromosome of Bacillus subtilis, and wequantitatively measured the resulting changes in the phenotypicnoise characteristics. We report that of these two parameters,increased translational efficiency is the predominantsource of increased phenotypic noise. This effect is consistentwith 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 geneErtugrul M. Ozbudak1, Mukund Thattai1, Iren Kurtser2, Alan D. Grossman2 & Alexander van Oudenaarden1Nature Genetics (2002)

http://www.nature.com/ng/journal/v31/n1/pdf/ng869.pdf

Figure 3. The burst size effect.

“working” noise e.g. in -phageIn some circumstances, noise can be highly desirable: an organism could use h

igh translation rates and large concentration fluctuationsas a means of creating nongenetic individuality in a population19,20.This is seen with the cI gene of λ-phage4,21: uponinfection of a host cell, the cI mRNA is transcribed with an efficientRBS upstream of the initiation codon, thus creating a highnoisestate; however, the lysogenic phenotype, once established,is maintained in a low-noise state (since transcription thenbegins at the initiation codon itself, producing inefficientlytranslated mRNA4).

More Background

E.coli statisticshttp://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi

About Diffusion in E.coli

Ratio of decay rates for different diffusion modes. Since thediffusion time is proportional to L2, long cells make higherdecay modes accessible to measurement. To obtain the ratio ofthe decay rates of the first and second Fourier modes on thesame cell, cells were treated with cephalexin, a drug whichinhibits 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 andthe cell center until GFP was completely photobleached. Thefirst and second Fourier modes were analyzed from recoverydata after photobleaching of the cell pole and center, respectively.An example of this experiment is presented in Fig. 1Eand F. Values obtained for Da were 7.2 6 1.3 mm2/s (average 6SD; n 5 8) for mode 1 and 6.8 6 1.2 mm2/s for mode 2,consistent with the experiments done without cephelaxin oncells 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