Team:Newcastle/SporulationTuning

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==Introduction==
==Introduction==
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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.
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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.<sup>[1]</sup> 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 [https://2009.igem.org/Team:Newcastle/Metalintakeefflux ‘metal container’], trapping the sequestered cadmium in its spore.
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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. More information about this other sub-project of ours can be found [https://2009.igem.org/Team:Newcastle/Chassis ''here''].
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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 [https://2009.igem.org/Team:Newcastle/Chassis chassis] comes into play, where the ''sleB'' and ''cwlJ'' germination-defective mutants are put into use.
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In order to control sporulation, our team is proposing the idea of inducing the synthesis of KinA, with IPTG as a sporulation initiation signal.  
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In order to control sporulation, our team proposed the idea of inducing the synthesis of ''kinA'', with IPTG as a sporulation initiation signal.  
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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,<sup>[1]</sup> which governs entry into the sporulation pathways of the bacterium Bacillus subtilis.<sup>[2]</sup>  
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[https://2009.igem.org/Team:Newcastle/SporulationTuning/Introduction#KinA ''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,<sup>[2]</sup> which governs entry into the sporulation pathways of the bacterium ''Bacillus subtilis''.<sup>[3]</sup>  
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:''[[Team:Newcastle/SporulationTuning/Introduction#Spo0A| ... Click to read more ...]]
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===Spo0A===
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<br>
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From our research, we found that entry into the sporulation pathway is governed by a member of the response regulator family of transcription facts, known as Spo0A.<sup>[2]</sup> Spo0A is activated by [https://2009.igem.org/Team:Newcastle/SporulationTuning/Phosphorylation phosphorylation] on an aspartyl residue located in the N-terminal portion of the protein.<sup>[2]</sup>
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Unlike other response regulators, Spo0A is indirectly phosphorylated by a multicomponent phosphorelay involving at least three kinases called KinA, KinB, and KinC, which phosphorylate Spo0F, and the resulting Spo0F~P, in turn, transfers the phosphoryl group to Spo0B. Finally, Spo0B~P transfers the phosphoryl group to, and thereby activates, Spo0A.<sup>[2]</sup> As such, the phophoryl groups are drained from the relay by the action of dedicated phosphotases that dephosphorylate Spo0F~P and Spo0A~P.<sup>[2]</sup>
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Spo0A is also subjected to control at the levels of its synthesis and activity by a positive feedback loop in which the response regulator stimulates the synthesis of the RNA polymerase σ factor σH, which, in turn stimulates transcription of the gene for Spo0A, as well as the genes for the phosphorelay components KinA and Spo0F.<sup>[1]</sup>
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It is important to note that activating Spo0A via the phosphorelay is essential as it is responsible for allowing Spo0A to accumulate in a gradual manner, and this slow accumulation plays a critical role in the ability of the regulatory protein to trigger sporulation.<sup>[2]</sup> Experiments have also been carried out and results have shown that the activated form of Spo0A failed to trigger sporulation during growth as it had bypassed phosphorylation, and only a small proportion of cells progressed to the early stage of sporulation.<sup>[2]</sup>
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===Sporulation===
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From our research, we found that phosphatases may be viewed as negative regulators that provide access for negative signals to influence the cell’s decision whether to sporulate or to continue vegetative growth.<sup>[3]</sup> The phosphotases that dephosphorylates Spo0A, preventing its activation are Spo0E, YisI, and YnzD.<sup>[4]</sup>
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Therefore, in order to ensure that sporulation occurs under the appropriate conditions, the phosphorelay must integrate the competition between signal input provided by the kinases and signal cancellation carried out by the phosphatises, which determines the decision to sporulate or not,<sup>[3][4]</sup> by governing flux through the relay and hence the level of Spo0A~P, which must reach a threshold concentration to trigger sporulation.<sup>[2]</sup>
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Other than the external and internal signals that are integrated into the phosphorelay, it is important to note that sporulation is also initiated by [https://2009.igem.org/Team:Newcastle/SporulationTuning/NutrientStarvation nutrient starvation], [https://2009.igem.org/Team:Newcastle/SporulationTuning/CellDensity cell density], and [https://2009.igem.org/Team:Newcastle/SporulationTuning/CellCycle cell cycle progression].<sup>[4]</sup>
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===KinA===
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Sporulation can be triggered with high efficiency in cells in the exponential phase of growth in rich medium by artificial induction of the synthesis of any one of three histidine kinases tha feed phosphoryl groups into the relay.<sup>[2]</sup> For our project, we will be using Kin A, a major histidine kinase responsible for activating the sporulation pathway in Bacillus subtilis.<sup>[5]</sup>
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KinA is a soluble cytoplasmic protein that appears to be active as a dimer and is composed of an amino-terminal sensor domain and a carboxy-terminal autokinase domain.<sup>[5]</sup>
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==Novelty in this sub-project==
==Novelty in this sub-project==
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Instead of allowing the cell to decide whether or not to sporulate, we hope to influence it's decision. We plan to use kinA as a part of this system.
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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.
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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 [https://2009.igem.org/Team:Newcastle/SporulationTuning#Modelling modelling section], with the Sporulation Tuning model controlling sporulation, and the Sin Operon model repressing sporulation, creating a more realistic model.
==Modelling==
==Modelling==
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The Sin (sporulation inhibition) Operon Model was one of the earlier models built.
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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.
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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.
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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.
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The Sin Operon and Sporulation Tuning model work hand in hand as components of the [https://2009.igem.org/Team:Newcastle/PopulationDynamics Population Dynamics model].
===KinA Expression Model===
===KinA Expression Model===
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Under normal conditions, LacI represses KinA.
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Under normal conditions, LacI represses ''kinA''.
[[Image:TeamNewcastleKinAExpLacIKinA.png|110px|center]]
[[Image:TeamNewcastleKinAExpLacIKinA.png|110px|center]]
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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
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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
[[Image:TeamNewcastleKinAExpLacIIPTG1.png|220px|center]]
[[Image:TeamNewcastleKinAExpLacIIPTG1.png|220px|center]]
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:''[[Team:Newcastle/Modelling/KinAExpression#KinA_Expression_Model| Click to view more of the KinA Expression Model equations]]''
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To understand more about the KinA Expression model, see [[Team:Newcastle/Modelling/SporulationTuning#KinA_Expression_Model]]
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<br>
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====Results====
====Results====
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The above equations were modelled in COPASI, and the following graphs show the behaviour of the system over time at different IPTG concentrations.
 
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[[Image:TeamNewcastleKinAExpPic1.png|center|300px]]
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;<u>IPTG concentration = 1000 nmol/fl</u>
 
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[[Image:TeamNewcastleKinAExpPic1.png|center|500px]]
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:''[[Team:Newcastle/Modelling/KinAExpression#Results| Click to view the KinA Expression results]]''
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<center>''Figure 1.1''</center>
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<br>
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In Figure 1.1, at a IPTG concentration of 1000nmol/fl, due to the high concentration of LacI*, it is difficult to view the results for the other species, such as, KinA, LacI, mRNA_KinA, and mRNA_LacI as they are all concentrated at the bottom.
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[[Image:TeamNewcastleKinAExpPic2.png|center|500px]]
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<center>''Figure 1.2''</center>
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Figure 1.2 shows the behaviour of KinA, LacI, mRNA_KinA and mRNA_LacI more clearly, at the IPTG concentration of 1000nmol/flu.
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From Figure 1.1 and 1.2, it is observed that the concentration of LacI* is significantly higher than the other species when the IPTG concentration is set at 1000nmol/fl. Looking at Figure 1.2, while the concentration of KinA is increasing, it is still lower than that of LacI.
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;<u>IPTG concentration = 3000 nmol/fl</u>
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[[Image:TeamNewcastleKinAExpPic3.png|center|500px]]
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<center>''Figure 2.1''</center>
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Comparing Figure 1.1 and 2.1, though the IPTG concentration in Figure 2.1 is 3000nmol/fl, the LacI* concentration is not particularly different.
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[[Image:TeamNewcastleKinAExpPic4.png|center|500px]]
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<center>''Figure 2.2''</center>
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As the IPTG concentration increases from 1000 to 3000nmol/fl, the KinA concentration is greater than the LacI concentration. However, upon a closer look, the KinA concentration did not increase due to the higher IPTG concentration. The KinA concentration is now higher than the LacI concentration because the LacI concentration decreased.
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;<u>IPTG concentration = 5000 nmol/fl</u>
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[[Image:TeamNewcastleKinAExpPic5.png|center|500px]]
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<center>''Figure 3.1''</center>
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Once again, as obsered in Figure 1.1 and 2.1, the concentration of LacI* in Figure 3.1 seems to reach the similar concentrations although the concentration of IPTG has been increased in each case. This seems to say that the concentration of LacI* has reached a threshold concentration.
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[[Image:TeamNewcastleKinAExpPic6.png|500px|center]]
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<center>''Figure 3.2''</center>
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The KinA concentration in Figure 3.2 is greater than that of LacI once again. However, KinA concentration seems to be stagnant at approximately 90nmol/fl, while the LacI concentration constantly decreases with increased IPTG concentration. There, the IPTG concentration only seems to affect the LacI concentration.
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===Sporulation Tuning Model===
===Sporulation Tuning Model===
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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.
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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.
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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'.<sup>[4]</sup>  
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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'.<sup>[5]</sup>  
The following equations describe the model:
The following equations describe the model:
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As Spo0F lacks an output domain and is incapable of activating transcription; it serves only as an intermediary in the phosphorelay. The phosphotransferase Spo0B transfers the phosphate from Spo0F~P to Spo0A.<sup>[4]</sup>
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:''[[Team:Newcastle/Modelling/SporulationTuning#Sporulation_Tuning_Model| Click to view more of the Sporulation Tuning Model equations]]''
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[[Image:TeamNewcastleSporeTuneEqn4.png|230px|center]]
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<center>''Equation 4''</center>
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[[Image:TeamNewcastleSporeTuneEqn5.png|160px|center]]
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<center>''Equation 5''</center>
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[[Image:TeamNewcastleSporeTuneEqn6.png|280px|center]]
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<center>''Equation 6''</center>
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[[Image:TeamNewcastleSporeTuneEqn7.png|160px|center]]
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<center>''Equation 7''</center>
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[[Image:TeamNewcastleSporeTuneEqn8.png|280px|center]]
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<center>''Equation 8''</center>
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[[Image:TeamNewcastleSporeTuneEqn9.png|160px|center]]
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<center>''Equation 9''</center>
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With respect to Equations 1 to 9, as seen above, the corresponding fluxes are as follows:
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[[Image:TeamNewcastleSporeTuneFluxes2.png|200px|center]]
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<center>''Fluxes''</center>
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The phophorylated proteins Spo0FP, Spo0BP and Spo0AP will go through degradation, and the following equations, Equation 10, 11 and 12 describes degradation.
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[[Image:TeamNewcastleSporeTuneEqn10.png|80px|center]]
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<center>''Equation 10''</center>
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[[Image:TeamNewcastleSporeTuneEqn11.png|80px|center]]
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<center>''Equation 11''</center>
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[[Image:TeamNewcastleSporeTuneEqn12.png|80px|center]]
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<center>''Equation 12''</center>
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<br>
====Results====
====Results====
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At a constant IPTG concentration of 5000nmol/fl, we vary the concentration of the sporulation signal, [SS] and the following graphs show the results.
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[[Image:TeamNewcastleSporeTunePic1.png|300px|center]]
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<u>'''At [SS] = 1000nmol/fl'''</u>
 
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[[Image:TeamNewcastleSporeTunePic1.png|500px|center]]
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:''[[Team:Newcastle/Modelling/SporulationTuning#Results| Click to view the Sporulation Tuning results]]''
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<center>''Figure 1.1''</center>
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Figure 1.1 shows that at an IPTG concentration of 5000nmol/fl and sporulation signal concentration of 1000nmol/fl, the Spo0F concentration gets significantly higher than the other components.
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===Sin (sporulation inhibition) Operon Model===
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In order to take a better look at the behaviour of the rest of the components, the following graphs were produced.
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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.
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[[Image:TeamNewcastleSporeTunePic2.png|500px|center]]
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<center>''Figure 1.2''</center>
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From Figure 1.2, the concentration of Spo0AP seems to be at a healthy concentration.
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[[Image:TeamNewcastleSporeTunePic3.png|500px|center]]
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<center>''Figure 1.3''</center>
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[[Image:TeamNewcastleSporeTunePic4.png|500px|center]]
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<center>''Figure 1.4''</center>
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<u>'''At [SS] = 3000nmol/fl'''</u>
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The concentration of the sporulation signal, [SS] is increased to 3000nmol/fl in the following graphs, and the results were investigated.
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[[Image:TeamNewcastleSporeTunePic5.png|center|500px]]
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<center>''Figure 2.1''</center>
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With a stronger sporulation signal, the concentration of Spo0FP and Spo0BP increased significantly, while the concentration of Spo0F dropped significantly from approximately 9000nmol/fl to 2300nmol/fl, comparing Figure 1.1 to 2.1.
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[[Image:TeamNewcastleSporeTunePic6.png|500px|center]]
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<center>''Figure 2.2''</center>
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The concentration of LacI* remained approximately the same, so as Spo0AP.
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[[Image:TeamNewcastleSporeTunePic7.png|center|500px]]
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<center>''Figure 2.3''</center>
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[[Image:TeamNewcastleSporeTunePic8.png|center|500px]]
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<center>''Figure 2.4''</center>
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Comparing Figure 1.3 and 2.4, the concentration of Spo0A falls from approximately 100nmol/fl to 50nmol/fl when [SS] is increased.
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Referring to the graphs obtained from the model, it seems like increasing the sporulation signal concentration, does not increase the Spo0AP concentration. However, the concentrations of Spo0BP and SpoFP did increase as the sporulation signal concentration increased.
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===Sin (sporulation inhibition) Operon Model===
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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.
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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.
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.
[[Image:TeamNewcastleSinOperonDiagram1.png|350px|center]]
[[Image:TeamNewcastleSinOperonDiagram1.png|350px|center]]
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<center>''Diagram 1: Simplifed Schematic of the sin Operon''<sup>[Reference]</sup></center>
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<center>''Diagram 1: Simplifed Schematic of the sin Operon''<sup>[9]</sup></center>
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The following differential equations makes up a deterministic model which describes the Sin Operon.
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:''[[Team:Newcastle/Modelling/SinOperon| Click to view more of the Sin Operon Model equations]]''
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[[Image:TeamNewcastleSinOperonEqn1.png|300px|center]]
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<center>''Equation 1: Concentration of SinI, [I]''</center>
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where:
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A<sub>I</sub>, the expression rate of SinI = 0.8 sec<sup>-1</sup>
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γ<sub>I</sub>, the degradation rate of SinI = 0.02 sec<sup>-1</sup>,
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k<sup>I</sup><sub>on</sub>, the rate of the formation of dimers = 0.083 nM<sup>-1</sup> sec<sup>-1</sup>,
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k<sup>I</sup><sub>off</sub>, the off rate for SinI:SinR = 0.5 sec<sup>-1</sup>,
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[[Image:TeamNewcastleSinOperonEqn2.png|300px|center]]
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<center>''Equation 2: Concentration of SinR, [R]''</center>
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where:
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A<sub>R</sub>, the expression rate of SinR = 0.014 sec<sup>-1</sup>
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γ<sub>R</sub>, the degradation rate of SinR = 0.002 sec<sup>-1</sup>
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k<sup>R</sup><sub>off</sub>, the off rate for SinR tetramers = 0.5 sec<sup>-1</sup>
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k<sup>R</sup><sub>on</sub>, the rate for tetramer formation = 0.00125 nM<sup>-3</sup> sec<sup>-1</sup>
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[[Image:TeamNewcastleSinOperonEqn3.png|150px|center]]
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<center>''Equation 3: Concentration of mRNA from P<sub>1</sub>, [m<sub>1</sub>]''</center>
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where:
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k<sub>1</sub>, the transcription rate from P<sub>1</sub> = 0.15 sec<sup>-1</sup>
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γ<sub>1</sub>, the degradation rate of m<sub>1</sub> = 0.005 sec<sup>-1</sup>
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[[Image:TeamNewcastleSinOperonEqn4.png|center|150px]]
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<center>''Equation 4: Concentration of mRNA from P<sub>3</sub>, [m<sub>3</sub>]</center>
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where:
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γ<sub>3</sub>, the degradation rate of m<sub>1</sub> = 0.005 sec<sup>-1</sup>
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[[Image:TeamNewcastleSinOperonEqn5.png|center|200px]]
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<center>''Equation 5: Concentration of SinR Tetramers, [R<sub>4</sub>]''</center>
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[[Image:TeamNewcastleSinOperonEqn6.png|center|300px]]
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<center>''Figure 6: Concentration of the SinI:SinR complexes, [IR]''</center>
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[[Image:TeamNewcastleSinOperonEqn7.png|450px|center]]
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<center>''Equation 7: Probability of P<sub>1</sub> in the Open Complex''</center>
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where:
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[S<sub>2</sub>] is the concentration of Spo0A~P dimers,
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[R<sub>4</sub>] is the concentration of SinR tetramers,
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[RNAP] = 30 nM is the concentration of free RNA polymerase available for transcription.
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The equilibrium association constants K<sub>i</sub>, where RT = 1.62, are defined as:
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K<sub>2</sub> = exp(-∆G<sub>2</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>2</sub> = -10.5
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K<sub>3</sub> = exp(-∆G<sub>3</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>3</sub> = -12.5
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K<sub>4</sub> = exp(-∆G<sub>4</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>4</sub> = -9.0
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K<sub>5</sub> = exp(-∆G<sub>5</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>5</sub> = -21.5
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K<sub>6</sub> = exp(-∆G<sub>6</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>6</sub> = -21.5
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K<sub>7</sub> = exp(-∆G<sub>7</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>7</sub> = -22.5
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K<sub>8</sub> = exp(-∆G<sub>8</sub>/RT) x 1e<sup>-9</sup>, where ∆G<sub>8</sub> = -33.5
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The Sin (sporulation inhibition) Operon Model is available for download as follows:
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* Text format: [[Media:TeamNewcastleSinOperonModel.txt]]
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<br>
====Results====
====Results====
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In the next few graphs, the concentration of SinR is varied to see how it affects the system.
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[[Image:TeamNewcastleSinOperonPicture1.png|200px|center]]
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[[Image:TeamNewcastleSinOperonPicture1.png|250px|center]]
 
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<center>''Figure 1.1''</center>
 
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:''[[Team:Newcastle/Modelling/SinOperon#Results| Click to view the Sin Operon Model results]]''
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[[Image:TeamNewcastleSinOperonPicture2.png|250px|center]]
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<br>
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<center>''Figure 1.2''</center>
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From Figure 1.1 and 1.2, which are essentially the same graphs, but at different time durations, it is observed that when the concentration of SinR is set at 1nM, the SinI concentration reaches a maximum of approximately 60nM.
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[[Image:TeamNewcastleSinOperonPicture3.png|250px|center]]
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<center>''Figure 2.1''</center>
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[[Image:TeamNewcastleSinOperonPicture4.png|250px|center]]
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<center>''Figure 2.2''</center>
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Referring to Figure 2.1 and 2.2, when the concentration of SinR is increased to 1.5, the concentration of SinI drops to approximately 50nM.
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[[Image:TeamNewcastleSinOperonPicture5.png|250px|center]]
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<center>''Figure 3.1''</center>
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[[Image:TeamNewcastleSinOperonPicture6.png|250px|center]]
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<center>''Figure 3.2''</center>
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When the concentration of SinR is set to 2nM, as seen in Figure 3.1 and 3.2, the concentration of SinI reaches a maximum of approximately 15nM.
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Comparing the previous results obtained, it seems like the concentration of SinI is most affected when the concentration of SinR is set at 2nM.
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[[Image:TeamNewcastleSinOperonPicture7.png|250px|center]]
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<center>''Figure 4.1''</center>
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[[Image:TeamNewcastleSinOperonPicture8.png|250px|center]]
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<center>''Figure 4.2''</center>
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The concentration of SinI further drops to 5nM when the concentration of SinR is increased to 2.5nM.
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The results from figures 1 to 4 shows that SinR does repress SinI. The greater the concentration of SinR, the lower the concentration of SinI. Also, the concentration of the SinR tetramers (R<sub>4</sub>) increases as well.
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As seen in Diagram 1, k<sub>3</sub> is a rate constant of P<sub>3</sub>. Therefore, at a low k<sub>3</sub> value, a high concentration of SinI would be expected. This theory is explored and the results are as follows:
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[[Image:TeamNewcastleSinOperonPicture9.png|250px|center]]
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<center>''Figure 5.1''</center>
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[[Image:TeamNewcastleSinOperonPicture10.png|250px|center]]
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<center>''Figure 5.2''</center>
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[[Image:TeamNewcastleSinOperonPicture11.png|250px|center]]
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<center>''Figure 5.3''</center>
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Figure 5.1, 5.2, and 5.3 justifies the theory, as it can be observed that the greater the k<sub>3</sub> rate constant, the lower the SinI concentration.
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The following graphs will investigate the effect on the system when the Spo0A protein concentration, [S<sub>2</sub>] is varied. The rate constants k<sub>3</sub> will be kept constant. The suggested range for [S<sub>2</sub>] is 1 to 10 000nM.
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[[Image:TeamNewcastleSinOperonPicture13.png|center|250px]]
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<center>''Figure 6.1''</center>
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[[Image:TeamNewcastleSinOperonPicture14.png|center|250px]]
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<center>''Figure 6.2''</center>
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[[Image:TeamNewcastleSinOperonPicture15.png|center|250px]]
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<center>''Figure 6.3''</center>
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From the above three figures, Figure 6.1, 6.2 and 6.3, the concentration of SinI increases as [S<sub>2</sub>] increases.
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==BioBrick constructs==
==BioBrick constructs==
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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.
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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'''
'''BBa_K174010'''
Line 462: Line 142:
==Lab Work Strategies==
==Lab Work Strategies==
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The lab work will mainly be to test our BioBrick using IPTG.
 
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===Construction===
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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).
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Synthesised by GeneArt, fragment:  2024bp
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[[Image:Newcastle KinA-sporulation1.JPG|center|500px]]
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[[Image:Newcastle KinA-sporulation1.JPG|center|400px]]
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<br>
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[[Image:Newcastle KinA-sporulation2.JPG|center|500px]]
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===Cloning===
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To find out more about our lab work strategies,
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The fragment will be returned from GeneArt cloned into pGFG-rrnB.
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:''see [[Team:Newcastle/SporulationTuning/Lab_Work_Strategies| Lab Work Strategies]]''
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Once the fragment is received from GeneArt  we need to clone it into a BioBrick compatible vector for submission to MIT. Digesting the fragment with EcoR1 and Pst1 and ligation to EcoR1 and Pst1 cut biobrick vector will do this.
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===Integration===
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==References==
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This is quite a difficult exercise because whilst pGFG-rrnB is designed to integrate at the amyE locus the presence of the native kinA on the 168 chromosome. The construct will preferentially integrate at kinA (disrupting it) rather than amyE. 
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A large number of transformants will need to be screened using starch plates and pcr to ensure that the correct mutants are selected that contain the construct integrated at only amyE.
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A backup strategy would be to integrate pMutin into the region immediately upstream of the native kinA ensuring that the native promoter and its control elements are preserved. The pSpac promoter would then be positioned upstream of kinA and therefore provide additional transcription in the presence of IPTG.
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===Testing and Characterisation===
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[1] Predich, M., Nair, G., Smith, I. (1992) ''Bacillus subtilis'' Early Sporulation Genes ''kinA'', ''spo0F'', and ''spo0A'' Are Transcribed by the RNA Polymerase Containing σ<sup>H</sup>. Journal of Bacteriology. Pp 2771-2778
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Once obtained the mutant will need to be tested for the induction of sporulation in the presence of IPTG.  
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This would involve microscopy and testing cultures for sporulation (e.g. by their heat resistance).
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[2] Veening, J-W., Smits, W. K., Kuipers, O. P. (2008) Bistability, Epigenetics, and Bet-Hedging in Bacteria. Annu. Rev. Microbiol. 62: 193-210
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[3] 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
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==References==
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[4] 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
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[1] Veening, J-W., Smits, W. K., Kuipers, O. P. (2008) Bistability, Epigenetics, and Bet-Hedging in Bacteria. Annu. Rev. Microbiol. 62: 193-210
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[5] 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
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[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
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[6] 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
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[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
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[7] Stephenson, K., Hoch, J. A. (2001) PAS-A domain of phosphorelay sensor kinase A: A catalytic ATP-binding domain involved in the initiation of development in ''Bacillus subtilis''. PNAS. Vol. 98, no. 26: 15251-15256
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[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
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[8] Veening, J-W., Hamoen, L. W., Kuipers, O. P. (2005) Phosphatases modulate the bistable sporulation gene expression pattern in ''Bacillus subtilis''. Molecular Microbiology 56(6), 1481-1494
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[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
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[9] Voigt, C. A., Wolf, D. M., Arkin, A. P. (2004) The ''Bacillus subtilis sin'' Operon: An Evolvable Network Motif. Genetics 169: 1187-1202
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{{:Team:Newcastle/Footer}}
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<br>
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{{:Team:Newcastle/Right}}
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Latest revision as of 02:11, 22 October 2009

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]

... Click to read more ...


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.

TeamNewcastleKinAExpLacIKinA.png


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

TeamNewcastleKinAExpLacIIPTG1.png


Click to view more of the KinA Expression Model equations


Results

TeamNewcastleKinAExpPic1.png


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:

TeamNewcastleSporeTuneEqn1.png
Equation 1


TeamNewcastleSporeTuneEqn2.png
Equation 2


TeamNewcastleSporeTuneEqn3.png
Equation 3


Click to view more of the Sporulation Tuning Model equations


Results

TeamNewcastleSporeTunePic1.png


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.

TeamNewcastleSinOperonDiagram1.png
Diagram 1: Simplifed Schematic of the sin Operon[9]


Click to view more of the Sin Operon Model equations


Results

TeamNewcastleSinOperonPicture1.png


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

TeamNewcastleBBKinA.jpg


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

TeamNewcastleBBKinAIPTG.jpg


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 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).

Newcastle KinA-sporulation1.JPG

To find out more about our lab work strategies,

see Lab Work Strategies

References

[1] Predich, M., Nair, G., Smith, I. (1992) Bacillus subtilis Early Sporulation Genes kinA, spo0F, and spo0A Are Transcribed by the RNA Polymerase Containing σH. Journal of Bacteriology. Pp 2771-2778

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

[3] 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

[4] 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

[5] 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

[6] 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

[7] Stephenson, K., Hoch, J. A. (2001) PAS-A domain of phosphorelay sensor kinase A: A catalytic ATP-binding domain involved in the initiation of development in Bacillus subtilis. PNAS. Vol. 98, no. 26: 15251-15256

[8] Veening, J-W., Hamoen, L. W., Kuipers, O. P. (2005) Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis. Molecular Microbiology 56(6), 1481-1494

[9] Voigt, C. A., Wolf, D. M., Arkin, A. P. (2004) The Bacillus subtilis sin Operon: An Evolvable Network Motif. Genetics 169: 1187-1202


Retrieved from "http://2009.igem.org/Team:Newcastle/SporulationTuning"