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Have a look at this link:
{{Imperial2010/Header}}
[http://www.openwetware.org/wiki/Imperial_College/Courses/Spring2008/Synthetic_Biology/Computer_Modelling_Practicals Synthetic Biology (Spring2008): Computer Modelling Practicals]
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<div class="accordionButton"><b>Objectives</b></div>
<div class="accordionContent",><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Objectives"><b>Here are our daily objectives.</b></a><br /><br /></div>


Have a look at Cell Designer to easily generate images of the system.
<div class="accordionButton"><b>Overview</b></div>
<div class="accordionContent",><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Overview"><b>Here is a short overview of the two models.</b></a><br /><br /></div>
<div class="accordionButton"><b>Output Amplification Model</b></div>
<div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten"><b>Model based on Michaelis Menten Kinetics</b></a><br />Comparison between different amplification models (HIV1 and TEV) based on Michaelis Menten kinetics. However, Michaelis Menten kinetics does not apply to our system. Therefore, it had to be modelled from first principle (see below using law of mass action).<br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten#HIV1"><img src="http://www.openwetware.org/images/0/03/Slide2.JPG" height="150" width="200" alt="Model using HIV1"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten#TEV"><img src="http://www.openwetware.org/images/4/48/TEV.jpg" height="150" width="200" alt="Model using TEV"/></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action"><b>Model based on Law of Mass Action</b></a><br />Comparison between these 3 different models: Simple production, 1-step and 2-step amplification.<br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_preA:_Simple_Production_of_Dioxygenase"><img src="http://www.openwetware.org/images/7/7f/Simple_production.JPG" height="100" width="300" alt="Simple Production"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_A:_Activation_of_Dioxygenase_by_TEV_enzyme"><img src="http://www.openwetware.org/images/1/1c/1-step_amplification.JPG" height="100" width="300" alt="1-step amplification"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_B:_Activation_of_Dioxygenase_by_TEV_or_activated_split_TEV_enzyme"><img src="http://www.openwetware.org/images/0/02/2-step_amplification.JPG" height="100" width="300" alt="2-step amplification"/></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables1"><b>Variables and Constants</b></a><br />Here are the variables and constants that are used in the Output Amplification Model.<br /><br /></div>
<div class="accordionButton"><b>Protein Display Model</b></div>
<div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Protein_Display"><b>Protein Display Model</b></a><br />This is a model showing when the ComD receptor will be activated (after proteins have been cleaved).<br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables2"><b>Variables and Constants</b></a><br />Here are the variables and constants that are used in the Protein Display Model.<br /><br /></div>
<div class="accordionButton"><b>Feedback from Wetlab</b></div>
<div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Experiments1"><b>Experiments for the Output Amplification Model</b></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Experiments2"><b>Experiments for the Protein Display Model</b></a><br /><br /></div>
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Example on how Valencia 2006 team used SimulLink to simulate their project: [http://www.igem.upv.es/igem06/images/7/7d/ECOLITASTER.ppt Valencia 2006 PowerPoint presentation]
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=Objectives=
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/>
===Week 6===
==Engineering approach to project==
*Get results for the output amplification models (hopefully, we can find constants; if not, we can say for which range of values our model will work)
[[Image:Engineering_cycle.jpg|500px|thumb|center]]
===Week 7===
1. Influence of Specification on Design and vice versa:
===Week 8===
*It was one of the longest steps as we were struggling to compromise the specifications with the viable designs.
===Week 9===
2. Influence of Design on Modelling and vice versa:
===Week 10===
*All modelling done was meant to give answers to questions that arose in the design phase.
=Output amplification model=
*Once designs were chosen, they were modelled. It was found that 2 step amplification is not likely to be effiecient, so it was decided that only 1step amplification will be taken forward to assembly. This was a significant conclusion as it would take weeks in the labroatory to find that out.
== First attempt==
*The doubt about big enough gradient of AIPs to be established in the extracellular space to set off receptor was rationalised by modelling. The model allowed to determine conditions for the system to work
 
3. Influence of Modelling on Assembly and vice versa:
Is it better to use TEV all the way or HIV1? Modelling should allows us to take decision which design is more efficient. If taken further, it will allow us to determine number of amplification steps that are most favourable.
*The results from modelling allowed to progress with assembly
 
4. Influence of Assembly on Testing and vice versa:
{|
*testing has been planned ahead, so assembly contructs have been modified to allow some testing methods like: purification or negative control.
| [[Image:Slide2.JPG|450px|thumb|center|alt=A|At each stage of amplification a distinct protease is being used ]]
5. Influence of Testing on Specifications and vice versa:
| [[Image:Model_output_011.jpg|450px|thumb|center|alt=A|At each stage of amplification a distinct protease is being used ]]
*We did not get there yet. However, if the specifications would not be met by the results, we would need to try redesigning the system or, in case of no alternative, changing the specifications.
|}
6. Influence of components not adjacent to each other in the cycle:
---------
*Testing may influence modelling as the results of the two do not match.
{|
*Many experiments were diesgned specifically on the request of modellers in order to find parameters for the models. Obtaining those paramters would increase the reliability of the models.
| [[Image:Slide1.JPG|450px|thumb|center|alt=A|TEV is used at both stages of amplification]]
| [[Image:Model_output_010.jpg|450px|thumb|center|alt=A|TEV is used at both stages of amplification]]
|}
 
== Second attempt ==
{|
| [[Image:Model_output_020.jpg.jpg|450px|thumb|center|alt=A|Model improved to account for the enzymes (protease action) ]]
|
|}
 
===Implementation in Matlab===
The Matlab code for the different stages of amplification and diagrams can be found [http://www.openwetware.org/wiki/Image:Modelling.docx here].
 
===Kinetic constants===
 
{| border="1"
! Quality
! GFP
! TEV
! split TEV
! split GFP
|-
|Km and Kcat
| Doesn't apply
|[http://peds.oxfordjournals.org/cgi/reprint/14/12/993 TEV constants (Km and kcat)]
| 40% of whole TEV
| Doesn't apply
|-
| half-life or degradation rate
| Half-life of GFP in Bacillus = 1.5 hours  - ref. Chris
| ?
| ?
| Half-life shorter than GFP
|-
| production rate in B.sub
| ?
| ?
| ?
| ?
|}
 
=== Conclusions ===
 
We couldn't obtain all the necessary constants. Hence, we decided to make educated guesses about possible relative values between the constants as well as varying them and observing the change in output.
 
As the result, we concluded that the amplification happens at each amplification level proposed. It's magnitude varies depending on the constants. There doesn’t seem to be much difference in substitution of TEV with HIV1.
 
==Modified version==
We cannot use Michaelis-Menten kinetics because of its preliminary assumptions, which our system does not fulfil.
These assumptions are:
*''Vmax is proportional to the overall concentration of the enzyme.''
But we are producing enzyme, so Vmax will change! Therefore, the conservation E0 = E + ES does not hold for our system.
 
*''Substrate >> Enzyme.''
Since we are producing both substrate and enzyme, we have roughly the same amount of substrate and enzyme.  
 
*''Enzyme affinity to substrate has to be high.''
 
Therefore, the model above is not representative of the enzymatic reaction. As we cannot use the Michaelis-Menten model we will have to solve from first principle (which just means writing down all of the biochemical equations and solving for these in Matlab).
 
===Production of Dioxygenase===
The reaction can be rewritten as:
TEV + split Dioxygenase <-> TEV-split Dioxygenase -> TEV + Dioxygenase.
This is a simple enzymatic reaction, where TEV is the enzyme, Dioxygenase the product and split Dioxygenase the substrate.
Choosing k1, k2, k3 as reaction constants, the reaction can be rewritten in these four sub-equations:
 
#[T'] = -k1[T][sD] + (k2+k3)[TsD] + sT - dT[T]
#[sD']= -k1[T][sD] + k2[TsD] + ssD - dsD[sD]
#[TsD'] = k1[T][sD] - (k2+k3)[TsD] - dTsD[TsD]
#[D'] = k3[TsD] - dD[D]
 
These four equations were implemented in Matlab, using a built-in function (ode45) which solves ordinary differential equations. The Matlab code for this module can be found [http://www.openwetware.org/wiki/Image:Matlab_Code_1.docx here].
[[Image:Fsd.jpg|450px|thumb|center|alt=A|Results of the Matlab simulation, setting all constants to 1]]
 
===Implementation in TinkerCell===
Another approach to model the amplification module would be to implement it in a program such as TinkerCell (or CellDesigner). It would also be useful to check whether the Matlab model works.
[[Image:Tinkercell.JPG|450px|thumb|center|alt=A|LHS: Network implemented in TinkerCell, RHS: constants and results ]]
 
 
 
===Improved model===
This version includes the following features:
*2 amplification steps (TEV and split TEV)
*Split TEV is specified to have a and b parts
*TEVa  is forbidden to interact TEVa (though in reality there could be some affinity between the two). Same for interaction between Tevb and Tevb
*Both TEV and TEVs are allowed to activate dioxugenase molecule
*Dioxugenase is assumed to be active as a monomer
*Activate split TEV (TEVs) is not allowed to activate sTEVa or sTEVb (this kind of interaction is accounted for in the next model version)
*There is no specific terms for time delays included
 
The MatLab code canbe found [http://openwetware.org/wiki/Image:Model_-_TEV-sTEV-sD_Code-diagram-results.docx here].
Note that no final conclusions should be drawn before realistic estimates for kinetic constants are included. It wasn’t done so far.
 
{|
| [[Image:ModelA.jpg|400px|thumb|center|alt=A|All chemical species appearing in the model]]
| [[Image:ModelA_(1).jpg|450px|thumb|center|alt=A|Network of the improved model]]
|-
| [[Image:ModelA_result1.jpg|450px|thumb|center|alt=A|Resulting graphs part 1. Compare the production graphs of TEV (transcribed and translated from scratch and the Dioxugenase which is the final species in the whole cascade]]
| [[Image:ModelA_result2.jpg|450px|thumb|center|alt=A|Resulting graphs part 2.]]
|}
 
===Further improvement===
This model is not implemented yet.
 
This version adds the following features:
* activated split TEV (TEVs) is allowed to activate not only sD but sTEVa and sTEVb
 
{|
| [[Image:ModelB_%281%29.jpg|450px|thumb|center|alt=A|Network of the further improved model]]
| [[Image:ModelB_%282%29.jpg|450px|thumb|center|alt=A|Network of the further improved model (continued)]]
|}

Latest revision as of 13:58, 12 October 2010

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} </style> </head> <body style="background-color:FFFFCC"> <div id="wrapper"> <div class="accordionButton"><b>Objectives</b></div> <div class="accordionContent",><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Objectives"><b>Here are our daily objectives.</b></a><br /><br /></div>

<div class="accordionButton"><b>Overview</b></div> <div class="accordionContent",><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Overview"><b>Here is a short overview of the two models.</b></a><br /><br /></div>

<div class="accordionButton"><b>Output Amplification Model</b></div> <div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten"><b>Model based on Michaelis Menten Kinetics</b></a><br />Comparison between different amplification models (HIV1 and TEV) based on Michaelis Menten kinetics. However, Michaelis Menten kinetics does not apply to our system. Therefore, it had to be modelled from first principle (see below using law of mass action).<br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten#HIV1"><img src="http://www.openwetware.org/images/0/03/Slide2.JPG" height="150" width="200" alt="Model using HIV1"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Michaelis_Menten#TEV"><img src="http://www.openwetware.org/images/4/48/TEV.jpg" height="150" width="200" alt="Model using TEV"/></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action"><b>Model based on Law of Mass Action</b></a><br />Comparison between these 3 different models: Simple production, 1-step and 2-step amplification.<br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_preA:_Simple_Production_of_Dioxygenase"><img src="http://www.openwetware.org/images/7/7f/Simple_production.JPG" height="100" width="300" alt="Simple Production"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_A:_Activation_of_Dioxygenase_by_TEV_enzyme"><img src="http://www.openwetware.org/images/1/1c/1-step_amplification.JPG" height="100" width="300" alt="1-step amplification"/></a> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Mass_Action#Model_B:_Activation_of_Dioxygenase_by_TEV_or_activated_split_TEV_enzyme"><img src="http://www.openwetware.org/images/0/02/2-step_amplification.JPG" height="100" width="300" alt="2-step amplification"/></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables1"><b>Variables and Constants</b></a><br />Here are the variables and constants that are used in the Output Amplification Model.<br /><br /></div>

<div class="accordionButton"><b>Protein Display Model</b></div> <div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Protein_Display"><b>Protein Display Model</b></a><br />This is a model showing when the ComD receptor will be activated (after proteins have been cleaved).<br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables2"><b>Variables and Constants</b></a><br />Here are the variables and constants that are used in the Protein Display Model.<br /><br /></div>

<div class="accordionButton"><b>Feedback from Wetlab</b></div> <div class="accordionContent"><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Experiments1"><b>Experiments for the Output Amplification Model</b></a><br /><br /><a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Experiments2"><b>Experiments for the Protein Display Model</b></a><br /><br /></div> </div>

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Engineering approach to project

1. Influence of Specification on Design and vice versa:

  • It was one of the longest steps as we were struggling to compromise the specifications with the viable designs.

2. Influence of Design on Modelling and vice versa:

  • All modelling done was meant to give answers to questions that arose in the design phase.
  • Once designs were chosen, they were modelled. It was found that 2 step amplification is not likely to be effiecient, so it was decided that only 1step amplification will be taken forward to assembly. This was a significant conclusion as it would take weeks in the labroatory to find that out.
  • The doubt about big enough gradient of AIPs to be established in the extracellular space to set off receptor was rationalised by modelling. The model allowed to determine conditions for the system to work

3. Influence of Modelling on Assembly and vice versa:

  • The results from modelling allowed to progress with assembly

4. Influence of Assembly on Testing and vice versa:

  • testing has been planned ahead, so assembly contructs have been modified to allow some testing methods like: purification or negative control.

5. Influence of Testing on Specifications and vice versa:

  • We did not get there yet. However, if the specifications would not be met by the results, we would need to try redesigning the system or, in case of no alternative, changing the specifications.

6. Influence of components not adjacent to each other in the cycle:

  • Testing may influence modelling as the results of the two do not match.
  • Many experiments were diesgned specifically on the request of modellers in order to find parameters for the models. Obtaining those paramters would increase the reliability of the models.
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