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Sporulation Tuning

Introduction

The bacteria, Bacillus subtilis, used in our project is a gram-positive soil bacterium that, under certain conditions, would commit itself to a developmental pathway leading to the production of spores.^[1] Therefore, in this section of our project, we hope to control sporulation in our bacterial population, such that we can decide how much of the population becomes spores, and how much continue as vegetative cells. Should the cell sporulate, it would become 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 as a result. Therefore, the role of chassis comes into play, where the sleB and cwlJ germination-defective mutants are put into use.

In order to control sporulation, our team is proposing 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 the 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]

... Read more ...

Novelty in this sub-project

In this sub-project, instead of allowing the cell to decide whether or not to sporulate, we hope to influence it's decision. In order to execute our plan, we intend to use the concentration of kinA, induced by IPTG to control sporulation.

Also, this sub-project consists of two main models, the Sporulation Tuning and Sin Operon model. 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 represses sporulation. This model was built to make our sporulation system a more realistic one.

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.

The Sin Operon and Sporulation Tuning model are meant to work hand in hand by integration into 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

To understand more about the KinA Expression model,

see KinA Expression Model Equations

Results

To view the results obtained from the KinA Expression Model,

see 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 with COPASI.

To proceed with the modelling of our Sporulation Tuning Model, we have decided that in response to an unidentified stimuli, where KinA autophosphorylates and then donates its phosphate groups to the response regulator Spo0F, the unidentified stimuli will be termed as 'sporulation signal'.^[4]

The following equations describe the model:

Equation 1

Equation 2

Equation 3

To understand more about the Sporulation Tuning Model,

see Sporulation Tuning Model Equations

Results

To view the results of the Sporulation Tuning Model,

see Sporulation Tuning Results

Sin (sporulation inhibition) Operon Model

In order to create a more realistic model of our sporulation system, in addition to the previous model, 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^[Reference]

To understand more about the Sin Operon Model,

see Sin Operon Model Equations

Results

To view the results obtained from the Sin Operon Model,

see 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

Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174010 here] for more information on this part.

BBa_K174011

IPTG inducable KinA sporulation trigger

Length: 1953bp

Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174011 here] for more information on this part.

Lab Work Strategies

The lab work executed would mainly be to induce sporulation in the presence of IPTG, and this would involve 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

References

[1] Veening, J-W., Smits, W. K., Kuipers, O. P. (2008) Bistability, Epigenetics, and Bet-Hedging in Bacteria. Annu. Rev. Microbiol. 62: 193-210

[2] Fujita, M., Losick, R. (2005) Evidence that Entry into Sporulation in Bacillus subtilis is Governed by a Gradual Increase in the Level and Activity of the Master Regulator Spo0A. 19: 2236–2244

[3] Sonenshein, A.L., Hoch, J.A., Losick, R., (2002) Bacillus subtilis and Its Closest Relatives From Genes to Cells. ASM Press, United States of America. Pp 476–477

[4] Hilbert, D.W., Piggot, P.J., (June 2004) Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation. Microbiology and Molecular Biology Reviews. Vol. 68, No. 2. Pp 234-262

[5] Eswaramoorthy, P., Guo, T., Fujita, M. (2009) In Vivo Domain-Based Functional Analysis of the Major Sporulation Sensor Kinase, KinA, in Bacillus subtilis. Journal of Bacteriology. Pp 5358-5368