Team:ETHZ Basel/Modeling/Interworking

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{{ETHZ_Basel10}}
 
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{{ETHZ_Basel10_Modeling}}
 
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= Evaluation for wet laboratory =
 
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[[Image:ETHZ_Basel_molecular_relation.png|thumb|400px|'''Combined models.''' Coupled individual models for the simulation of the whole process and their interfaces. The concentration of CheYp determines the movement bias.]]
 
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== Goals of the evaluation ==
 
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To determine the best possible parts for the biological implementation of E. lemming, the combined molecular model (Light switch - Chemotaxis) has been used to answer specific questions:
 
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# Which receptor (Che) protein should be attacked?
 
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# To which light-sensitive protein (PhyB, PIF3) should the receptor protein be linked?
 
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# What is a good aspartate concentration?
 
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# In what quantity should the anchor and binding light-sensitive protein be chosen?
 
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Answers to this questions were able to decrease the experimental expenses significantly.
 
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== Evaluator ==
 
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[[Image:ETHZ_Basel_models_evaluator_amplitude.png|thumb|400px|'''Determination of relative amplitude.''' Relative amplitude of free CheYp was maximized to determine optimal parts.]]
 
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Evaluation of the results is a general optimization problem. The output variable in the case of E. lemming is the concentration of free and phosphorylated CheYp, because it is directly linked to the movement bias. To achieve a reasonable evaluation variable, the relative amplitude of free and phosphorylated CheYp was chosen to be maximized.
 
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Because of the limited amount of different combination possibilities, it was decided to evaluate all combinations. Parameter space of variable experimental implementation possibilities is shown in Table 1.
 
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{| border="1" align="center"
 
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|+ '''Table 1: Experimental parameters'''
 
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! Che !! LSP !! LSP' !! [Asp] !! [AP] !! [anchor] !! model
 
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|-
 
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| CheR || PhyB || PIF3 || 0 uM || 0 uM || 0 uM || Spiro
 
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|-
 
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| CheB || PIF3 || PhyB || 10^-6 uM || 25 uM || 25 uM || Mello
 
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|-
 
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| CheY |||||| 10^-3 uM || 50 uM || 50 uM ||
 
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|-
 
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| CheZ |||||||| 75 uM || 75 uM ||
 
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|-
 
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||||||||| 100 uM || 100 uM ||
 
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|}
 
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The questions were answered in a hierarchical order, e.g. try Che1 before Che2.
 
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== Results ==
 
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=== 1. Which receptor (Che) protein should be attacked? ===
 
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{| border="0" align="center"
 
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|- valign="top"
 
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|[[Image:ETHZ_Basel_models_evaluator_cher.png|thumb|200px|'''Che = CheR.''' CheR has A = 0.25.<br>(CheR, LSP:PIF3, Asp:Medium, Model:Spiro)]]
 
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|[[Image:ETHZ_Basel_models_evaluator_cheb.png|thumb|200px|'''Che = CheB.''' Inverse relation of activation / inactivation of CheB limit possible applications in E. lemming. A = 0.16.<br>(CheB, LSP:PIF3, Asp:Medium, Model:Spiro)]]
 
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|[[Image:ETHZ_Basel_models_evaluator_chey.png|thumb|200px|'''Che = CheY.''' Candidate protein CheYp has A = 0.8.<br>(CheY, LSP:PIF3, Asp:Medium, Model:Spiro)]]
 
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|[[Image:ETHZ_Basel_models_evaluator_chez.png|thumb|200px|'''Che = CheZ.''' CheZ showed the most increase in terms of relative amplitude. A = 1.4.<br>(CheZ, LSP:PIF3, Asp:Medium, Model:Spiro)]]
 
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|}
 
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Although CheZ showed the highest relative amplitude, it was not chosen to be the first candidate, since it is not strictly located at the cell membrane and therefore would possibly not be suitable for our method of deactivation by changing local amount of active protein by spatial dislocation. The second best candidate in terms of relative amplitude was CheY, which was chosen to be the first target. CheR and CheB would have been possible backup targets in this order.
 
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In addition, CheB and CheZ showed inverse activation / inactivation behavior than CheR and CheY upon light pulse induction. This means, that the activity would increase and therefore, this would have to be corrected by increasing the concentration of the proteins far above wild-type level to achieve an inverse effect of activation.
 
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=== 2. To which light-sensitive protein (PhyB, PIF3) should the receptor protein be linked? ===
 
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{| border="0" align="center"
 
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|- valign="top"
 
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|[[Image:ETHZ_Basel_models_evaluator_phyb.png|thumb|400px|'''LSP? = PhyB.''' PhyB coupling is very robust and results in maximal plateau surface.]]
 
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|[[Image:ETHZ_Basel_models_evaluator_pif3.png|thumb|400px|'''LSP? = PIF3.''' Coupling to PIF3 results in a much more sensitive area of [AP] vs. [anchor].]]
 
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|}
 
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PhyB is according to the evaluation the more robust choice compared to PIF3, but PhyB has a tendency to sequestering, which results in a lowered active concentration of the species, because the proteins stick together. In addition, PIF3 is much smaller than PhyB and it could therefore have a lower negative influence on the Che protein. Relative amplitude of both combinations is very similar. For this reasons, PIF3 was the first choice as light-sensitive protein linked to the Che protein.
 
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=== 3. What is a good aspartate concentration? ===
 
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{| border="0" align="center"
 
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|- valign="top"
 
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|[[Image:ETHZ_Basel_models_evaluator_asp0.png|thumb|260px|'''[Asp] = 0.''' No aspartate in the systems results in very low relative amplitude. A = 0.03.]]
 
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|[[Image:ETHZ_Basel_models_evaluator_asp1.png|thumb|260px|'''[Asp] = 10^-6.''' Medium aspartate levels show reasonable amplitude levels of A = 0.3.]]
 
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|[[Image:ETHZ_Basel_models_evaluator_asp2.png|thumb|260px|'''[Asp] = 10^-3.''' High aspartate levels reach saturation of receptors and cannot increase the relative amplitude further.]]
 
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|}
 
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Aspartate concentration should be chosen to reach saturation or near-saturation of aspartate receptor. In this example, medium aspartate levels were able to reach near-saturation of the receptors and therefore, this parameter value was chosen.
 
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=== 4. In what quantity should the anchor and binding light-sensitive protein be chosen? ===
 
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According to the the evaluation of all 48 parameter combination possibilities, it was chosen to take a concentration for anchor plasmid of [anchor] = 50 uM and a concentration for the anchor binding protein [AP] = 40 - 50 uM, which is slightly below to ensure having enough binding positions.
 
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=== Comparison of the chemotaxis models ===
 
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Both chemotaxis receptor models implemented in the combined model predicted very similar results. An obvious difference is the missing consideration of CheZ in the Mello model.
 
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= Evaluation for information processing =
 
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== Goal of the evaluation ==
 
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Main goal of the evaluation for information processing was to determine time constants of E. lemming by using the combined molecular model (Light switch - Chemotaxis).
 
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== Evaluator ==
 
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[[Image:ETHZ_Basel_models_evaluator_ip.png|thumb|400px|'''Evaluation of activation / inactivation time constants.''' Time elapsed upon activation of the system until the amplitude reached 2^-(1/2) of the local maximum was measured.]]
 
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Again, the output variable was the concentration of free and phosphorylated CheYp. To determine the time constants, the time elapsed upon activation of the system until the amplitude reached 2^-(1/2) of the local maximum was measured.
 
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== Results ==
 

Latest revision as of 12:13, 19 October 2010