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Stochastic Switch

Introduction

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.

Novelty in this sub-project

We intend to design a synthetic stochastic switch by using an invertible segment of our DNA that codes a promoter. Depending on the direction of the promoter, coding sequiences will be expressed which reflect the decision to be a metal container or not. We also plan to nudge the natural stochasticity of the sporulation system towards greater sporulation rates, for this we intend to use Spo0A phosphorylation.

Modelling

Stochastic Modelling Tools

=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= is another stochastic simulation tool which also uses Gillespie’s direct method and supports SBML. CellML model for the expression of Hin system

Media:flipping.txt

We have used computational modelling in Matlab to try to determine how to make our system tuneable.

Please see our modelling page for Matlab files on our stochastic switch model.

BioBrick constructs

Metal Container Decision

Our stochastic switch decides whether the spores can germinate, or whether they are commited to be a metal containing spore that cannot germinate again. We need this switch as we cannot interrupt the natural life cycle of the bacteria, as a proportion have to go on to seed the next generation.

We looked at the following possibilities:

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

The Hin system will be the main DNA rearrangement system within our stochastic switch, Unlike the fim system the hin system allows the DNA segment to be flipped back and forth, therefore a pulse of hin expression is what we would need to ensure we can get the correct proportion to be metal containers.

The switch as an overall diagram Media:MetalContainerDecisionSwitch.ppt

Animation of how the switch works

Media:switch animation.ppt

Lab Work Strategies

Other Presentations and Diagrams

fimE switch

The FimE switch is a similar switch to the Hix system, however it acts as a latch, meaning once flipped, the segmant will not flip back.

1. 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. Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping
2. 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...

Bistability in Bacillus subtilis

Read this page to find more options for natural stochastic switches in Bacillus subtilis. Natural stochastic switches:Bistability in Bacillus subtilis

Spo0A phosphorylation

Spo0A phoshorylation controls sporulation and is also a bitsable switch. 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