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Contents 1 Stochasticity 1.1 Hin/Hix system 1.2 fimE switch 1.2.1 The search for fimE equivalent in Bacillus subtilis 1.3 Spo0A phosphorylation 1.3.1 Sin operon details 1.4 Stochastic Modelling Tools 1.4.1 Matlab 1.4.2 Stocks 2

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.

Natural stochastic switches:Bistability in Bacillus subtilis

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

1. [http://genomics.lbl.gov/Stuff/TimHam-BandB-online%20version.pdf 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)

Organism |No. | Gene |Bp | Putative Function | Score | E-value

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

1. [http://jb.asm.org/cgi/reprint/183/14/4190?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=subtilis&searchid=1&FIRSTINDEX=880&resourcetype=HWFIG Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping]
2. [http://ukpmc.ac.uk/articlerender.cgi?artid=310841 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...

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.

1. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1449569
2. http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2430929&blobtype=pdf
3. http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.micro.62.081307.163002

Sin operon details

[http://www.ncbi.nlm.nih.gov/nuccore/143546?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum#sequence_143546 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

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.