Team:Newcastle/SporulationTuning

=Sporulation Tuning=

Introduction
The bacterium Bacillus subtilis used in our project is a gram-positive soil bacterium which, under certain conditions, commits itself to a developmental pathway leading to the production of spores.[1] In this part of our project, we aim to control sporulation in our bacterial population, so that we can decide how much of the population becomes spores, and how much continue as vegetative cells. Should the cell sporulate, it becomes a ‘metal container’, trapping the sequestered cadmium in its spore.

After the cell sequesters cadmium into its spore, it should not germinate, or the sequestered cadmium will be released back into the environment. Therefore, the chassis comes into play, where the sleB and cwlJ germination-defective mutants are put into use.

In order to control sporulation, our team proposed the idea of inducing the synthesis of kinA, with IPTG as a sporulation initiation signal.

KinA is a major kinase which provides phosphate input to a phosphorelay, which in turn, activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor,[2] which governs entry into the sporulation pathways of the bacterium Bacillus subtilis.[3]


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Novelty in this sub-project
In this sub-project, instead of allowing the cell to decide whether or not to sporulate, we influence its decision. In order to execute our plan, we used the concentration of kinA, induced by IPTG, to control sporulation.

This sub-project consists of two main models: the Sporulation Tuning and the Sin Operon models. These two models are meant to work together, as mentioned below in the modelling section, with the Sporulation Tuning model controlling sporulation, and the Sin Operon model repressing sporulation, creating a more realistic model.

Modelling
The Sin (sporulation inhibition) Operon Model was one of the earlier models built. As its name suggests, it models the repression of sporulation. The Sin Operon Model was built in CellML.

The second model built was the simple model of KinA expression. After satisfactory results were obtained, the sporulation phosphorelay was modelled into the KinA Expression Model, and is known as the Sporulation Tuning Model. Both the KinA Expression and Sporulation Tuning models were built using COPASI.

The Sin Operon and Sporulation Tuning model work hand in hand as components of the Population Dynamics model.

KinA Expression Model
Under normal conditions, LacI represses kinA.



However, in the presence of IPTG, KinA can be expressed, as IPTG binds to lacI, deactivating it. Equations (a) and (b) describes how IPTG binds to LacI, forming LacI*, which is the deactivated form of LacI


 *  Click to view more of the KinA Expression Model equations

Results



 *  Click to view the KinA Expression results

Sporulation Tuning Model
The expression of KinA has been modelled as seen above, therefore we can now proceed further into the Sporulation Tuning Model, which is built from the KinA Expression Model using COPASI.

To proceed with the modelling of our Sporulation Tuning Model, we have decided that in response to an unidentified stimulus, where KinA autophosphorylates and then donates its phosphate groups to the response regulator Spo0F, the unidentified stimuli will be termed as 'sporulation signal'.[5]

The following equations describe the model:

Equation 1

Equation 2

Equation 3


 *  Click to view more of the Sporulation Tuning Model equations

Results



 *  Click to view the Sporulation Tuning results

Sin (sporulation inhibition) Operon Model
In order to create a more realistic model of our sporulation system the team has decided to include the Sin (sporulation inhibition) Operon Model, which the team designed in CellML.

The sin operon controls the production and activity of the repressor SinR, which in its active tetrameric form, inhibits sporulation by repressing stage II and spo0A promoters. On the other hand, the accumulation of Spo0A~P induces the expression of SinI, which binds to and inactivates SinR.

Diagram 1: Simplifed Schematic of the sin Operon[9]


 *  Click to view more of the Sin Operon Model equations

Results



 *  Click to view the Sin Operon Model results

BioBrick constructs
The BioBrick we have designed is to contain an IPTG inducable kinA gene, using pSpac, allowing us to test the theory about KinA in the lab.

BBa_K174010

KinA

Length: 1818bp



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BBa_K174011

IPTG inducable KinA sporulation trigger

Length: 1953bp



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Lab Work Strategies
The lab work executed was be to induce sporulation in the presence of IPTG, and this involved microscopy and testing cultures for sporulation (e.g. by their heat resistance).



To find out more about our lab work strategies,
 * see Lab Work Strategies