IGEM:IMPERIAL/2007/Cell By Date/Design: Difference between revisions

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<li>[[IGEM:IMPERIAL/2007/Cell By Date/Fabrication|Fabrication]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Fabrication|Fabrication]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Testing|Testing]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Testing|Testing]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/DataAnalysis|DataAnalysis]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Validation|Validation]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Validation|Validation]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Notes|Notes]]</li>
<li>[[IGEM:IMPERIAL/2007/Cell By Date/Notes|Notes]]</li>
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This is the main page for Cell By Date 2 this page will be split up into various sections in the future.
==Overview of Design==


==1. Our Target : Beef==
[[Image:CellByDateDesignWiki.png]]


We all know that beef goes off. The dominant organisms leading to the spoilage of beef depend of the beef's composition and the environmental conditions under which the beef is stored. For refrigerated packaged beef Pseudomonas spp. were dominate areobically while Lactobacillus was dominant anaerobically. [[IGEM:IMPERIAL/2007/Cell By Date/References|(12.Labuza,1993)]]
{|
|-
!Property:
!Value
!Design Solution
!System Level
|-
|Health Regulations
|System Must not be living replicating bacteria
|Use a Cell Free System e.g. Promega's S30 Cell Extract
|Chassis
|-
|Lifespan
|System must have a shelf life of 7 days
|Protease Inhibitor of Cell Extract Should ensure degradation of Visual Reporter is Minimal<br> Proper Packaging should ensure that evaporation of Cell Free system is so low that <br> system can surive for 7 days
|Chassis
|-
|Inputs
|Isothermal Conditions between 0 & 40 C
|Exploit Thermal Dependance of rates of expression
|Construct
|-
|
|Dynamic conditions eg. steps & ramps
|                                                "
|Construct
|-
|Outputs
|System should give a visual signal <br> when beef is off
|Couple constituitive promoter to a Fluoresent Protein eg. RFP
|Construct
|-
|Activation Energy
|System Needs to have an activation Energy 30 +/- kJ/mol
|To be Determined - this is hard to design for
|Construct
|-
|Response Time
|System needs to have a response time under 1 hour
|To be Determined - this is hard to design for
|
|}


There also seems to be a general rule for beef that when the bacterial count reaches 10^7cm^-2 , off odours and slime production occour and the beef is considered off. [[IGEM:IMPERIAL/2007/Cell By Date/References|(13.Food Hygiene,Microbiology and HACCP)&(10.Leak,1999)]]
==Chassis Selection==


The Gompertz's model is widely used when considering beef spoilage as it has been shown to fit growth data very well[[IGEM:IMPERIAL/2007/Cell By Date/References|(12.Labuza,1993)]].  Using the Gompertz model we can get the specific growth rate, Lag phase duration (LPD) and maximum population density (MPD) for bacterial growth at a particular constant temperature.  And then using these we can determine the Activation Energy Ea, for the beef spoilage rxn.  For U2 grade Argentinian beef stored in polyethylene and SARAN PVC the Ea ranged from 80kJmol^-1 to 220kJmol^-1 for a range of bacteria. [[IGEM:IMPERIAL/2007/Cell By Date/References|(11.Giannuzzi,1997)]]
We have chosen to use the commerciall available S30 extract made by Promega.  After having looking into a variety of different chassis here (link to cell free section) we feel that this chassis best suits our needs. In particular this chassis allows us to meet our base requirement of compling with the Health and Safety regulations of the field we are working in, we don't want a live system near our burger meat as potential leak of our system could mean that our system actually spoils the burger meat !!


One contary value for the Ea of the beef spoilage rxns is given by Leak [[IGEM:IMPERIAL/2007/Cell By Date/References|(10.Leak,1999)]] who calculated Ea=30kJmol^1.  The difference between Leak's value and that of Giannuzzi probably lies in their packaging methods, i do not know Leak's packaging methods so this is just a guess.
In addition to complying with health regulations the S30 cell extract is commercially available meaning that it has been shown to work.  This is very important for us as it allows our focus to be on tuning the chassis to suit our needs rather than making the chassis work in the first place.


==2. Our System : pTET mimicing a TTI==
==Construct Selection==


Several Technologies have already been developed to addressed the problem of monitoring the heat exposure of products in the cold chain.  One particular family of these products are called Temperature Time Integrators (TTI).[[IGEM:IMPERIAL/2007/Cell By Date/References|(2.Labuza,2006)]]
In previous research and also in previous iGEM project, Temperature as an input has been explored through cold shock and heat shock promoters.  These promoters essentially only operatre for a given range of temperatures.


The key aspect of a TTI is that they are based on a phenomenon which can act as a signal to a consumer for example, eg. a colour change.  The rate at which this change occours needs to be temperature dependant so it can mimic the effect temperature has on the spoiling of meat eg. change happens quicker at higher temperatures.  In order for a TTI to accurate report the spoilage rxn of beef the activation energy of the two rxns needs to be similar.  For example a difference between the two Ea's less than 20kJmol^-1 would result in the TTI estimating the thermal history of the beef to be within 1 degree C of the actual history.).[[IGEM:IMPERIAL/2007/Cell By Date/References|(1.Taoukis,2006)]]
In making a Time Temperature Integrator we would like a promoter that works at all temperatures, increasing its rate of protein synthesis as temperature increases.  We can realise this behaviour by using a simple constituitive promoter and exploiting the thermal dependance of its rate of synthesis, this type of behaviour has been characterised by Ryals as far back as 1982.


I have tried to calculate the Ea of our system, pTET linked to Mut3BGFP, and have got a value of 1.5kJmol^-1.  If we take the Leak's Ea of the spoilage rxn to be 30kJmol^-1 then our TTI will be accurate to 2 Degrees.  However if we take the Giannuzzi's highest value of 220kJmol^-1 and assuming Taoukis estimation rule to be linear, our TTI will be accurate to 10 degrees C of the thermal history of the beef.
In terms of Activaiton Energy and Response Time we have been unable to find these in literature and to it is hard to make a design that will achieve these targets.  However through the course of our experimentation we will determine these properties and hopefullly their values will suit our interests.

Latest revision as of 10:22, 19 October 2007

Cell by Date


Overview of Design

Property: Value Design Solution System Level
Health Regulations System Must not be living replicating bacteria Use a Cell Free System e.g. Promega's S30 Cell Extract Chassis
Lifespan System must have a shelf life of 7 days Protease Inhibitor of Cell Extract Should ensure degradation of Visual Reporter is Minimal
Proper Packaging should ensure that evaporation of Cell Free system is so low that
system can surive for 7 days 		Chassis
Inputs Isothermal Conditions between 0 & 40 C Exploit Thermal Dependance of rates of expression Construct
Dynamic conditions eg. steps & ramps " Construct
Outputs System should give a visual signal
when beef is off 		Couple constituitive promoter to a Fluoresent Protein eg. RFP Construct
Activation Energy System Needs to have an activation Energy 30 +/- kJ/mol To be Determined - this is hard to design for Construct
Response Time System needs to have a response time under 1 hour To be Determined - this is hard to design for

Chassis Selection

We have chosen to use the commerciall available S30 extract made by Promega. After having looking into a variety of different chassis here (link to cell free section) we feel that this chassis best suits our needs. In particular this chassis allows us to meet our base requirement of compling with the Health and Safety regulations of the field we are working in, we don't want a live system near our burger meat as potential leak of our system could mean that our system actually spoils the burger meat !!

In addition to complying with health regulations the S30 cell extract is commercially available meaning that it has been shown to work. This is very important for us as it allows our focus to be on tuning the chassis to suit our needs rather than making the chassis work in the first place.

Construct Selection

In previous research and also in previous iGEM project, Temperature as an input has been explored through cold shock and heat shock promoters. These promoters essentially only operatre for a given range of temperatures.

In making a Time Temperature Integrator we would like a promoter that works at all temperatures, increasing its rate of protein synthesis as temperature increases. We can realise this behaviour by using a simple constituitive promoter and exploiting the thermal dependance of its rate of synthesis, this type of behaviour has been characterised by Ryals as far back as 1982.

In terms of Activaiton Energy and Response Time we have been unable to find these in literature and to it is hard to make a design that will achieve these targets. However through the course of our experimentation we will determine these properties and hopefullly their values will suit our interests.

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