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Meetings Calendar

Stochasticity

One of the most unique aspects of our project is our synthetic stochastic switch which regulates the decision to be a metal container spore, or a spore that can go on to germinate as part of the normal life cycle.

Stochastic Switch Examples

Natural switches:Bistability in Bacillus subtilis

SinR and SinI can be used to regulate the heavy metal sequestration. Only in a sub population of the bacteria, sinI will be expressed at sufficient levels to trigger our system. Whereas sinR will be expressed in all cells. So with this approach we can say that only a small population of the cells will get the heavy metals. If we want the other way we can switch the roles of SinI and sinR.With the normal conditions only 2% of the population express enough sinI.[2]

Spo0A phosphorylation

Spo0A phoshorylation itself is also a bitsable switch so that a subset of the population will be on spo0A ON state [2,3]. We can trigger sporulation therefore by expressing more KinA on one side of the switch.

Sin operon details

Sin operon in GenBank

sinR: Positive regulation of comK; negative regulation of aprE, kinB, sigD, spo0A, spoIIA, spoIIE, spoIIG

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000964.2&from=2551885&to=2552220&dopt=gb

sinI:Antagonist of SinR

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000964.2&from=2551678&to=2551851&dopt=gb

Hin/Hix system

In 2006, Davidson team tried to solve the burnt pancake problem by using DNA rearrangement using Hin/Hix system from Salmonella typhimurium. (http://parts2.mit.edu/wiki/index.php/Davidson_2006.) Basically they tried to use the bacteria as a biomemory! They also have a paper published which is attached.

Their animation explains the process quite well. (http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf)

The parts they submitted to the parts registry have "W" flag which means they are working. http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2006&group=iGEM2006_Davidson

fimE switch

fimE switch for DNA re-arrangement

A Tightly Regulated Inducible Expression System Utilising the fim Inversion Recombination Switch.(E. Coli) Timothy S. Ham, Sung Kuk Lee, Jay D. Keasling,Adam P. Arkin,Received 21 December 2005; accepted 2 March 2006 Published online 13 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20916

We could use it switch off or on the production of a protein of our choice, such as the genes involved in germination.

The search for fimE equivalent in Bacillus subtilis

Gene list from BLAST search output in Subtilist web-server (7 matches)

B.subtilist|BS000101011332|RipX: 295 site specific integrase... 82 | 2.00E-17

B.subtilist|BS000101010965|CodV: 303 sitespecific integrase... 73 | 1.00E-14

B.subtilist|BS000101012099|YdcL: 367 unknown; similar to int... 30 | 0.13

B.subtilist|BS000101011909|AraM: 393 L arabinose operon 27 | 0.64

B.subtilist|BS000101013550|YoeC: 94 unknown; similar to unkn... 25 | 3.2

B.subtilist|BS000101013687|YorC: 125 unknown 24 | 7.1

B.subtilist|BS000101011532|IlvD: 557 dihydroxyacid dehydratase 24 | 7.1

B.subtilist|BS000101010010|YybT: 658 unknown similar to unk... 23 | 9.2

Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping
Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the sigma K-encoding gene =>...DNA rearrangement that depends on the spoIVCA gene product...

Stochastic Modelling Tools

Matlab

Matlab can be used for stochastic modelling. Glasgow team used Matlab implementing Gillespie algorithm to incorporate noise among cells. They also used deterministic modelling using ODEs and compared their results. When the number of cells increase two approaches become similar since the noise is cancelled out.

Stocks 2

Stocks 2 is another stochastic simulation tool which also uses Gillespie’s direct method and supports SBML.

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

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 ==Stochastic Switch Examples==
 ===[[Team:Newcastle/ Bistability in B.Subtilis|Natural switches:Bistability in ''Bacillus subtilis'']]===
-The ''Sin'' (Sporulation Inhibition) operon can be used as the stochastic switch. It is a natural bistable switch.
-[[Image:SinOperon.jpg]]
-During normal conditions, ''SinR'' is expressed constitutively by P3 and keeps its concentration at a constant level repressing promoter 1. When transcription of promoter1 is activated by phosphorylated Spo0A, both sinI and sinR are expressed from promoter 1. ''SinI'' inactivates ''SinR'' by forming a complex with ''sinR'' upregulating both proteins from promoter1. This cross repression, inhibition of SinR by ''SinI''  and  the transcriptional represion of sinI by ''SinR'', forms the basis of the bistability. While the positive feedback in the production of ''SinI'' enhances the bistability, it also causes increase in ''sinR'' levels because of the expression from promoter 1 hence causing oscillations.[1]
-The ''Sin'' operon controls the early stages of sporulation and has a key role to control the sporulation without disturbing its regulation. The threshold of this switch to progress into sporulation can be controlled by varying some parameters, hence providing population heterogeneity.  Tight binding of ''SinR'' to the first promoter region  with fewer ''sinR'' molecules increases this heterogeneity. By mutating the first promoter, the binding affinity of sinR the promoter can be increased.
-With variations on ''SinI'':''SinR'' interaction strength,  transcription rate of ''sinR'' from the third promoter and the expression rate of ''SinR'', dynamics of the system can be altered.[1]
 ''SinR'' and ''SinI'' can be used to regulate the heavy metal sequestration. Only in a sub population of the bacteria, sinI will be expressed at sufficient levels to trigger our system. Whereas sinR will be expressed in all cells.

Team:Newcastle/Stochasticity