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Meetings Calendar

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. Whilst stochastic oscillators have been implemented before using transcriptional regulators, our switch makes use of an invertable DNA segment to ensure that the decision is heritable.

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 sequences will be expressed which reflect the decision to be a metal container or not. We also plan to tune the natural stochasticity of the sporulation system towards greater sporulation rates by altering the rate Spo0A phosphorylation.

BioBrick constructs

There will be various bricks involved within the stochastic switch construct, both those involved in the complete system as well as those designed for testing within the lab. The stochastic brick construct uses the Hin invertase system in order to flip a promoter region between Hix sites. The directionality of the promoter determines whether the switch is 'on' or 'off'. When the promoter is facing right it allows transcription of genes that control:

- Prevention of germination
- Upregulation of sporulation rate
- Expression of the metal sponge (SmtA)
- Decreased cadmium efflux
- Upregulation of cadmium import

The following diagram shows our stochastic construct:

Prevention of germination

The prevention of germination is governed by another invertase switch. Once the stochastic promoter faces right, a FimE protein is expressed which inverts a further promoter region. This promoter controls expression of the CwlD and SleB genes knocked out within our chassis. If their promoter is in the correct orientation then the cell will be able to germinate and continue as a vegetative cell. However if their promoter has been flipped, the cell can not germinate following sporulation, and will be trapped as a metal containing spore.

Upregulation of sporulation rate

The upregulation of sporulation involves increasing KinA expression. kinA codes a kinase protein that phosphorylates the Spo0A protein to its active form. When the promoter region within our stochastic brick faces right, there will be increased KinA expression, and thus a greater sporulation rate.

Metal sponge and cadmium influx/efflux

Expression of the metallothionein fusion protein (CotC-GFP-SmtA), cadmium import channel (mntH) and the cadmium efflux channel (CadA) is also governed by the direction of the stochastic promoter. When the direction of promoter faces right, metallothionein fusion protein's expression will be triggered to soak the cadmium. While the import channel is upregulated, the efflux system's activity will be slowed down to increase the number of cadmium inside the cell.

Stochastic Brick

We have decided to get our stochastic construct synthesised, as trying to build the construct manually would be too time costly. The following sequencher diagram shows the components of the construct we had synthesised.

Testing construct

In order to test our construct we have had to redesign using inducible promoters governing Hin invertase expression. We have used promoters pSpac and pxylA (Induced by IPTG and Xylose) to test our system. We include cut sites around these promoters in order to replace them with SigmaA promoters once the construct has been characterised.(See sequencher diagram above)

Degradation controller

In order to have another level of control over the orientation of the promoter within the flipping region we have added a degradation tag to the hin invertase protein. The following paper describes how proteins including modified ssrA tags can be located to the ClpXP protease by an Sspb protein. This means that inducible Sspb expression can requlate degradation levels of the tagged protein.

[http://www3.interscience.wiley.com/journal/121415079/abstract?CRETRY=1&SRETRY=0 Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP ]

We have decided to put the Sspb protein under the control of an arabinose inducible promoter as the following diagram illustrates, also we have included a region of the sac gene in our construct, so that the region will integrate into the Bacillus genome at a region other than amyE.

We added a modified version of ssrA degradation tag onto the C-terminus of Hin protein. Hence expressed proteins are degraded by ClpXP. However mutations on the ssrA tag weaken the recognition by ClpX and the modified tags require SspB adaptor protein to be recognized. When SspB protein is expressed, the proteins tagged with modified version of ssrA tag are targeted for degradation, otherwise they remain stable.

In B. subtilis there is no sspB orthologue and SspB from E. coli works in B. subtilis. By regulating the levels of SspB by arabinose, we designed an inducable protein degradation device.

Wild type E. coli ssrA tag is AANDENY-ALAA (SspB recognition site – ClpX recognition site). As suggested in the paper, we took one of the modified ssrA tags to use in our system.

AANDENY-SENY-ALGG (SspB recognition site – SENY +4 Linker - ClpX recognition site)

This tag works well in B. subtilis however degradation tags can affect activity of proteins. Different degradation tags may have effect on the activity of different proteins. It has been shown that this tag effected the activity of ComA(1).

Griffith, K. L., and A. D. Grossman. 2008. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70:1012-1025.

We have the following cloning strategies for testing our construct.

Lab Work Strategies

Cloning strategies

Currently in the lab we are half way to acheiving a complete manual brick which is the degradation controller. In the lab so far, the stochastic switch team have:

Rehydrated, transformed, and miniprepped the biiobricks involved in promoter replacement, as well as frozen down these E.coli strains into the TPA collection.
Prepared the pSB1AT3 plasmid backbone (restriction digest, gel extraction) ready for ligation.
Have successful PCR products for the sac region (bacillus) and sspb (E.coli), and a possible success for the ara region.
Have done digests for sspb and sac, sspb has been extracted from the gel and is due for cloning.

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.

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

Team:Newcastle/Stochasticity