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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==
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
[[Image:Engineering_cycle.jpg|500px|thumb|center]]
|-
1. Influence of Specification on Design and vice versa:
  ! Day !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday !! Weekend
*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:
  | Date || 09 || 10 || 11 || 12 || 13 || 14-15
*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.
  | Objective ||  || ||  || Find constants
*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
| Find protein production constants and TEV reaction rate constants ||
3. Influence of Modelling on Assembly and vice versa:
|-
*The results from modelling allowed to progress with assembly
  | Completion || || || ||We didn't manage to complete the task || The orders of magnitude established - ready to run simulations ||
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:
===Week 7===
*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.
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
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.
  ! Day !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday !! Weekend
*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.
|-
  | Date || 16 || 17 || 18 || 19 || 20 || 21-22
|-
  | Objective || Implementing the constant ranges in the output model. Comparing the results between the models. || Start modelling the protein display signalling to find the concentrations ||  || || ||
|-
  | Completion || || || || || ||
|}
===Week 8===
===Week 9===
===Week 10===
 
=Output amplification model=
Motivation: We have come up with a simple concept of amplification of output done by enzymes. Before the final constructs are assembled within the bacterial ogranism, it is beneficial for us to model the behaviour of our design.
 
The questions to be answered:
# How beneficial use of amplification is? (compare speeds of response of transcription based output to amplified outputs)
# How many 'amplification levels' are beneficial to have? (if too many amplification steps are involved, the associated time delay with expressing even amplfiied output may prove it not to be beneficial.
# Does mixing of amplfication levels have a negative infleunce on output? Is it better to use TEV all the way or HIV1? Modelling should allows us to take decision which design is more efficient.
 
== First attempt==
 
{|
| [[Image:Slide2.JPG|450px|thumb|center|alt=A|At each stage of amplification a distinct protease is being used ]]
| [[Image:Model_output_011.jpg|450px|thumb|center|alt=A|At each stage of amplification a distinct protease is being used ]]
|}
---------
{|
| [[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==
===Michaelis Menten kineticsdoes not apply===
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).
===Changes in the system===
GFP is not used any more as an output. It is dioxygenase acting on the catechol (activating it into colourful form). Catechol will be added to bacteria, it won't be produced by them. Hence, basically in our models dioxynase is going to be treated as an output as this enzyme is recognised as the only activator of catechol in our system. This means that change of catechol into colourful form is dependent on dioxygenase concentration.
=== Models: ===
====Model preA: Production of Dioxygenase====
This model includes transcription and translation of the dioxygenase.
It does not involve any amplification steps. It is our control model against which we will be comparing the results of other models.
 
====Model A: Activation of Dioxygenase by TEV enzyme====
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 ]]
 
 
 
====Model B: Activation of Dioxygenase by TEV or activated split TEV enzyme====
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.]]
|}
 
=====Model C: 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)]]
|}
 
===Constants for Model===
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
|-
  ! Type of constant !! Derivation of value
|-
  | TEV Enzyme dynamics
  | Enzymatic Reaction:
E + S <-> ES -> E + P
 
Let
*k1 = rate constant for E + S -> ES
*k2 = rate constant for E + S <- ES
*kcat = rate constant for ES -> E + P
 
We know that Km = (kcat + k2)/k1
Assuming that kcat << k2 << k1, we can rewrite Km ~ k2/k1
 
From this [http://peds.oxfordjournals.org/cgi/reprint/14/12/993 paper] constants for TEV can be found:
*e.g. wildtype TEV
*Km = 0.062 +/- 0.010 mM
*kcat = 0.16 +/- 0.01 s^-1
 
These values correspond with our assumption that kcat ~ 0.1 s^-1 and Km ~ 0.01 mM.
 
Hence, we can estimate the following orders of magnitude for the rate constants:
*k1 ~ 10^5 M^-1 s^-1
*k2 ~ 10^3 s^-1
 
Using these values should be a good approximation for our model.
|-
  | Degradation rate
(common for all)
  | Assumption: To be approximated by cell division (dilution of media) as none of the proteins are involved in any active degradation pathways
Growth rate (divisions/h): [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC235685/pdf/jbacter00326-0019.pdf 0.53<G.r.<2.18]
 
Hence on average,  g.r.=1.5 divisions per hour
=> 1 division every 1/1.5=0.6667 of an hour (40mins)
 
To deduce degradation rate use the following formula:
 
τ_(1⁄2)=ln2/k
 
Where τ_(1⁄2)=0.667 hour, k – is the degradation rate
 
k=ln2/τ_(1⁄2) = 0.000289 s^(-1)
 
|-
  | Production rate
 
(TEV and dioxygenase)
  |
We had real trouble finding the production rate values in the literature and we hope to be able to perform experiments to obtain those values for (TEV protease and catechol 2,3-dioxygenase). The experiments will not be possible to be carried out soon, so for the time being we suggest very simplistic approach for estimating production rates.
 
We have found production rates for two arbitrary proteins in E.Coli. We want to get estimates of production rates by comparing the lengths of the proteins (number of amino-acids).
 
As this approach is very vague, it is important to realise its limitations and inconsistencies:
*Found values are taken from E.Coli not B.sub.
*The two found rates are of the same value for quite different amino-acid number which indicates that protein folding is limiting the production rates (we use the chosen approach as the only way of getting the estimate of order of reaction)
 
 
LacY production = [http://bionumbers.hms.harvard.edu/bionumber.aspx?s=y&id=100738&ver=0&hlid=29205 100 molecules/min]  ([http://www.uniprot.org/uniprot/P02920 417 Amino Acids])
 
LacZ production = [http://bionumbers.hms.harvard.edu/bionumber.aspx?s=y&id=100737&ver=0&hlid=29206 100 molecules/min] ([http://www.uniprot.org/uniprot/P00722 1024 AA])
 
Average production ≈ 100molecules/min 720 AA
 
That gives us:
*TEV production ≈ 24 molecules/min = 0.40 molecules/s ([http://www.uniprot.org/uniprot/P04517 3054 AA])
 
As production rate needs to be expressed in concentration units per unit volume, the above number is converted to mols/s and divided by the cell volume → 2.3808*10^(-10) mol*dm^(-3)*s^(-1)
*C23D production ≈ 252 molecules/min = 4.2 molecules/s ([http://www.uniprot.org/uniprot/P54721#section_x-ref 285 AA]) → 2.4998*10^(-9) mol*dm^(-3)*s^(-1)
 
We will treat these numbers as guiding us in terms of range of orders of magnitudes. We will try to run our models for variety of values and determine system’s limitations.
|-
|Kinetic parameters
of dioxygenase
|
Initial velocity of the enzymatic reaction was investigated at pH 7.5 and 30 °C.
*Wild type (we use that one)
 
Km = 10μM
 
kcat = 52 s^(−1)
*Mutated type
 
Km = 40μM
 
kcat = 192 s^(−1)
 
Consequently, the kcat/Km 4.8 of the mutant was slightly lower than kcat/Km 5.2 of the wild type, indicating that the mutation has little effect on catalytic efficiency.
 
[http://www.springerlink.com/content/e3718758m5052214/fulltext.pdf reference]
|-
|Dimensions of
Bacillus subtillis cell
|
Dimensions of Basillus subtilis (cylinder/rod shape) in reach media (ref. bionumbers):
# diameter: d= 0.87um
# length: l=4.7 um in rich media
This gives:  Volume= π∙(d/2)^2∙l=2.793999 μm^3≈ 2.79∙10^(-15) dm^3 
 
 
|}

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    <ul class='plan'>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Strategy' class='listmain' id='plan'>Strategy</a></li>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Parts/Synthesis' class='listmain' id='plan'>Synthesis</a></li>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Lab_Work' class='listmain' id='plan'>Lab Work</a></li>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Protocol' class='listmain' id='plan'>Protocol</a></li>
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      <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Modelling' class='listmain' id='plan'>Modelling</a></li>
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     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Journal_Club' class='listmain' id='extra'>Journal Club</a></li>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Movie_Night' class='listmain' id='extra'>Movie Night</a></li>
     <li><a href='http://openwetware.org/wiki/IGEM:Imperial/2010/Judging_Criteria' class='listmain' id='extra'>Judging Criteria</a></li>
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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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