IGEM:IMPERIAL/2007/Projects/Cell by date/Modelling: Difference between revisions

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==Summary:==


In the design section we outlined which variants of Cell By Date we would like to implement in different Chassis, as shown in the table below. <br>


{| style="background:#FAF5FF; border:1px solid #FFDAB9; color:#000000"
==Overview of Modelling ==
|- style="background:#FFA07A"
 
|| '''Chassis''' || Level 1 || Level 2 || Level 3
Welcome to our portal page for the modelling of Cell By Date
|- style="background:#FFDAB9"
 
|style="background:#FFA07A" |  E.coli || X || || ||  
Cell by date tries to improve on printed cell by date as acting as a thermal exposure device, exploiting the thermal dependance of the rate of expression of a simple reporter system.  We are looking at a variety of constructs to realise this behaviour.
|- style="background:#FFDAB9"
 
| style="background:#FFA07A" | In-Vitro|| X || X || X ||
===Constructs to be Used ===
|- style="background:#FFDAB9"
 
| style="background:#FFA07A" | In-Veso|| X || X || X ||
{|
|-
|GFP Based
|[[IMAGE:ICGEMS_dvc_Ptet_gfp.png|left|frame]]
|[[IMAGE:ICGEMS_dvc_PT7_gfp.png|left|frame]]
|[[IMAGE:PcI_construct.PNG|left|frame]]
|-
|RFP Based
|[[IMAGE:ICGEMS_dvc_Ptet_rfp.PNG|left|frame]]
|[[IMAGE:ICGEMS_dvc_PT7_rfp.PNG|left|frame]]
|[[IMAGE:ICGEMS_dvc_PcI_rfp.PNG|left|frame]]
|-
|}
|}


In this section we modify the variants of Cell by Date so that they can be implemented advantageously in each chassis.  We also model these modified variants in order to understand the behaviour we expect or to tune the system to realise a particular behaviour.  This leads us to write up experiments to determine if our system behaves the way our models predict.
As can be seen in the above table all of our constructs have the same general form of a promoter upstream of a reporter.  Looking at the rate of Fluorescent Protein (FP) produced :
 
<math>\frac{d[FP]}{dt}=k_{FP}(t)-d_{FP}[FP] </math>


==Overview of Modelling ==
# kFP : Function of Temperature.  k is based on the promoter used as promoters take time to turn on.
# dFP : Function of System.  May be considered to be a function of temperature as proteins degrade faster at higher temperatures.


Overall our modelling for this project will take the form of
{| cellspacing="5"
|-valign="top"
|width=50%|


<math>\frac{dFP}{dt}=k_{FP}(t)-d_{FP}[GFP] </math>


# kFP : Function of Temperature.  k is based on the promoter used as promoters take time to turn on.
<!--ROW 2-->
# dFP : Function of System.  May be considered to be a function of temperature as proteins may degrade at high temperatures.
|-valign="top"
||


===Transient Response of System===


With this in mind we will look at two graphs of k vs. time (special pt is kON.) and [FP] vs. time key point is [FP]ss
Image needs to be changed to matlab plot of system


* Our major problem at the moment is estimating the errosrs involved with our fluorometer and Pipette we hope to address these through calibration curves.
[[Image:ConstantTempCBDresponseC.png|left|500px]]<br clear = "all">


For each experiment we will do the following
||


#Calibration curve to determine error in Fluorometer
===Transfer Function of System===
#Decay Experiment @ Varying temperatures
#Plug Together to find transient response and k
#Find these parameters as a function of Temperature


===Construct Specific modelling===
Image needs to be changed to matlab plot of system


====For all constitutive promoters (Ptet promoter used as an example): ====
[[Image: CellByDate_tf.jpg|thumb|left|500px|Construct 2 - LuxR not expressed]]<br clear = "all">


[[IMAGE:ICGEMS_dvc_Ptet_gfp.png|left|frame|Picture5]]<br clear = "all">
|}


1.Apply the above general equation to this specific construct.
===Feedback from Protocols/"Manufacturing"===


<math>\frac{d[GFP]}{dt}=k_{Ptet}(t)-d_{GFP}[GFP] </math>
====Construct to be pursued====
*pTet           


2.calibration curve to determine error in Fluorometer - we are trying to get this infromation from BertholdTech <br>
Refer to the [[IGEM:IMPERIAL/2007/DNA_Constructs|constructs]] page for more information.
3.Decay Experiment at varying temperatues:
*This is to determine <math>d_{GFP}</math>
*The Fluroresence of a pure sample of GFP kept at 'constant temperature' will be measured at time intervals. We expect the fluroesence to decay exponentially with time.
*We plan to determine the decay constant in the following way:
**We cannot measure the decay constant directly, instead we plan to measure determine the half-life of our reporter.  With this done we can calculate the decay constant using the following equation:
<math>t_{1/2} = \frac{\ln 2}{\lambda}</math> <br>
[http://en.wikipedia.org/wiki/Exponential_decay DecayPage]


4.Having found <math>d_{GFP}</math> we look at transient respone of construct at several constant temperatuers to find k at constant temperature<br>
===Performance Specifications===
5.Find k & d as functions of temperature:
*This will hopefully have been carried out by interpolation of data from 4. and 3. eg. plots of k vs. temp & d vs. temp <br>
**The problem with this method is that will not allow us to determine the response time of the promoters eg. the time taken for the promoter to respond to a temperature change.
Otherwise another plan is as follows:
*This plan has have several temperature slopes as inputs from which we can compare changes in temperature with changes in k to determine the relationship between the two.<br>
**This methods involes comparing rate of temp increase to rate of k change eg. substituting time in k expression for Temp.<br>
6.Following on from this we plan to have several pulse inputs, similar to what we'll have in real life eg. taking meat from supermarket and bringing it back home to put in your fridge.  From this we can work out how well our system perfroms in real life scenarios, and how well our model performs.

Latest revision as of 08:56, 30 August 2007

Cell by Date: Modelling




Overview of Modelling

Welcome to our portal page for the modelling of Cell By Date

Cell by date tries to improve on printed cell by date as acting as a thermal exposure device, exploiting the thermal dependance of the rate of expression of a simple reporter system. We are looking at a variety of constructs to realise this behaviour.

Constructs to be Used

GFP Based
RFP Based

As can be seen in the above table all of our constructs have the same general form of a promoter upstream of a reporter. Looking at the rate of Fluorescent Protein (FP) produced :

[math]\displaystyle{ \frac{d[FP]}{dt}=k_{FP}(t)-d_{FP}[FP] }[/math]

  1. kFP : Function of Temperature. k is based on the promoter used as promoters take time to turn on.
  2. dFP : Function of System. May be considered to be a function of temperature as proteins degrade faster at higher temperatures.


Transient Response of System

Image needs to be changed to matlab plot of system


Transfer Function of System

Image needs to be changed to matlab plot of system

Construct 2 - LuxR not expressed

Feedback from Protocols/"Manufacturing"

Construct to be pursued

  • pTet

Refer to the constructs page for more information.

Performance Specifications

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