1. miscellaneous… & iGEM Design V1.0 - Xiuye 13.6.2008

2. 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)

3. Original project proposal Part 1: random#-generator Options of Part 2 (read-out)  Simple read-out (positive feedback?)  Combination of spatial independent #s  Combination of temporal independent #s All monitored by fluorescence

4. Part 1 Design ideas By (manually?) capturing phases in oscillation… Fluctuations in Random Diffusion etc… Identical promoters…mutually exclusive?

5. Collins’ Toggle-switch

6. 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

7. 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. MoreMore Michael B. Elowitz, Ph.D. California Institute of Technology Pasadena, CA http://www.hhmi.org/news/elowitz_bio.html

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

9. “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

10. 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

11. Figure 3. The burst size effect.

12. “working” noise e.g. in -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).

13. More Background E.coli statistics http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi About Diffusion in E.coli

14. 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

17. Margolin, 2006 CurBio

18. Shih et al, PNAS 2003

19. Huitema et al, 2006 Cell