Team:Tokyo Tech/Genetic toggle switch to implement rewritable function

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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="85%" align="center"
!align="center"|[[Team:Tokyo_Tech|Home]]
!align="center"|[[Team:Tokyo_Tech|Home]]
!align="center"|[[Team:Tokyo_Tech/Construction|Construction]]
!align="center"|[[Team:Tokyo_Tech/Construction|Construction]]
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!align="center"|[[Team:Tokyo_Tech/Genetic toggle switch to implement rewritable function|Genetic toggle switch ]]
!align="center"|[[Team:Tokyo_Tech/Genetic toggle switch to implement rewritable function|Genetic toggle switch ]]
!align="center"|[[Team:Tokyo_Tech/Parts|Parts Submitted to the Registry]]
!align="center"|[[Team:Tokyo_Tech/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Tokyo_Tech/Result|Result]]
!align="center"|[[Team:Tokyo_Tech/Team|The Team]]
!align="center"|[[Team:Tokyo_Tech/Team|The Team]]
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!align="center"|[[Team:Tokyo_Tech/Notebook|Notebook]]
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!align="center"|[[Team:Tokyo_Tech/Acknowledgements|Acknowledgements]]
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= Genetic toggle switch to implement rewritable function=
= Genetic toggle switch to implement rewritable function=
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We think two type models - genetic circuit model and mathematical model.
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In order to implement rewritable function, we tried to construct Pressure Inducible Genetic Toggle Switch (in short PIGTS).
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We thought two types of model - genetic circuit model and mathematical model - so as to construct PIGTS.
== Genetic circuit model ==
== Genetic circuit model ==
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[[Image:Toggle_genetic_circuit_model.png|frame|center|fig5-1:genetic circuit model]]
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[[Image:Toggle_genetic_circuit_model.png|thumb|400px|right|figure 5-1 genetic circuit model]]
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'''1. Writable function'''
'''1. Writable function'''
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* Plac is induced under 30MPa pressure(PL is weakly induced)
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* '''Plac''' is induced under 30MPa pressure  
* Express CI and GFP  
* Express CI and GFP  
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* CI represses PL and causes low LacI concentration
* CI represses PL and causes low LacI concentration
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* Express increasingly CI and GFP ⇒ '''Bright!!'''
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* Express increasingly CI and GFP('''inserted destruction tags''') ⇒ '''Bright!!'''
'''2. Erasable function'''
'''2. Erasable function'''
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* PL is induced by heat
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* '''PL''' is induced by heat
* Express LacI and LacI represses Plac  
* Express LacI and LacI represses Plac  
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== Mathematical model ==
== Mathematical model ==
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[[Image:Room_pressure_model.png|frame|right|fig5-2:Room pressure model]]
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<gallery widths="200px" heights="150px" perrow="1" align="right" caption="Differential equation models">
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[[Image:30MPa_pressure_model.png|frame|right|fig5-3:30MPa pressure model]]
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Image:Room_pressure_model.png|figure 5-2 Atmospheric pressure model. It is known that n<sub>P<sub>L</sub></sub> = 3.0 from earlier studies
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Image:30MPa_pressure_model2.png|figure 5-3 30MPa pressure model
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</gallery>
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We have to choose promoter-RBS strength which satisfy following condition to implement rewritable function.
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# Converge to high CI concentration state under 30MPa (writable)
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# Bistable under ordinary pressure (erasable)
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[[Image:Desirable_condition.png|frame|left|fig5-4:Desirable condition]]
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We used two differential equation models and we call them 'atmospheric pressure model' and '30MPa pressure model'.
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In order to implement rewritable function, we decided to use the difference of nullclines of each system so as to satisfy the following condition
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* Converge to high CI concentration state under 30MPa pressure (writable)
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* Bistable under atmospheric pressure (erasable)
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[[Image:Desirable_condition.png|700px|thumb|left|figure 5-4 Desirable condition<!--. left:under atmospheric pressure (nullclines of figure 5-2), right:under 30MPa pressure (nullclines of figure 5-3).-->]]
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{{clear}}
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=== IPTG assay ===
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[[Image:Transfer_Function.png|thumb|300px|left|figure 1 Hill function]]
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'''Objective'''
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By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.
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'''Result & Conclusion'''
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GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in figure 1 and the formulation of Hill function fitting described above, the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained '''n<sub>P<sub>lac</sub></sub> = 2.15'''.
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{{clear}}
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== Calculate the domain of appropriate parameters ==
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[[Image:Desirable_condition_94.png|400px|thumb|right|figure 5-5 The domain of appropriate parameters if &alpha;<sub>Plac</sub> = 3.0]]
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It is known that Plac activity under 30MPa pressure is '''94.0''' times stronger than Plac activity under atmospheric pressure, so, '''&beta;<sub>30P<sub>lac</sub></sub>''' = 94.0.
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If 30MPa additional activity rate of PL ('''&beta;<sub>30P<sub>L</sub></sub>''') or the ratio of Plac - RBS strength ('''&alpha;<sub>P<sub>lac</sub></sub>''') and PL-RBS strength('''&alpha;<sub>P<sub>L</sub></sub>''') are not appropriate value, we can't construct PIGTS.
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So we calculated the domain of appropriate &beta;<sub>30P<sub>L</sub></sub> and the ratio of &alpha;<sub>P<sub>lac</sub></sub> and &alpha;<sub>P<sub>L</sub></sub>.
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== Desirable condition ==
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[[Image:PL_pressure_response.png‎|200px|none|thumb|left|figure 5-6 Pressure-response ability of PL]]
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We identified '''&beta;<sub>30P<sub>L</sub></sub> = 1.42''' by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose '''P<sub>L</sub> - RBS strength range from 2.4 to 7.4'''.

Latest revision as of 14:22, 26 October 2008

Home Construction Acrylic container Development of promoter Genetic toggle switch Parts Submitted to the Registry Result The Team Acknowledgements


Contents

Genetic toggle switch to implement rewritable function

In order to implement rewritable function, we tried to construct Pressure Inducible Genetic Toggle Switch (in short PIGTS). We thought two types of model - genetic circuit model and mathematical model - so as to construct PIGTS.

Genetic circuit model

figure 5-1 genetic circuit model

1. Writable function

  • Plac is induced under 30MPa pressure
  • Express CI and GFP
  • CI represses PL and causes low LacI concentration
  • Express increasingly CI and GFP(inserted destruction tags) ⇒ Bright!!


2. Erasable function

  • PL is induced by heat
  • Express LacI and LacI represses Plac
  • Therefore, GFP expression decreases

Mathematical model

Differential equation models

figure 5-2 Atmospheric pressure model. It is known that nPL = 3.0 from earlier studies

figure 5-3 30MPa pressure model

We used two differential equation models and we call them 'atmospheric pressure model' and '30MPa pressure model'. In order to implement rewritable function, we decided to use the difference of nullclines of each system so as to satisfy the following condition

  • Converge to high CI concentration state under 30MPa pressure (writable)
  • Bistable under atmospheric pressure (erasable)


figure 5-4 Desirable condition

IPTG assay

figure 1 Hill function

Objective

By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.

Result & Conclusion

GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in figure 1 and the formulation of Hill function fitting described above, the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained nPlac = 2.15.

Calculate the domain of appropriate parameters

figure 5-5 The domain of appropriate parameters if αPlac = 3.0

It is known that Plac activity under 30MPa pressure is 94.0 times stronger than Plac activity under atmospheric pressure, so, β30Plac = 94.0. If 30MPa additional activity rate of PL (β30PL) or the ratio of Plac - RBS strength (αPlac) and PL-RBS strength(αPL) are not appropriate value, we can't construct PIGTS. So we calculated the domain of appropriate β30PL and the ratio of αPlac and αPL.

figure 5-6 Pressure-response ability of PL

We identified β30PL = 1.42 by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose PL - RBS strength range from 2.4 to 7.4.

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