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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==Overview of Design==


[[Image:CellByDateDesignWiki.png]]


==3. End Result and what has to be done yet==
{|
|-
!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
|
|}


The deliverables of this project pertain to how effective our system is as a TTI for hamburger meat.
==Chassis Selection==


One deliverable of this project would be a specification sheet showing how well our system works in a variety of scenarios.  By looking at the various plots I would like on this specification sheet I can determine what needs to be done in the next few weeks.  Firstly I would like to generate a isothermal 'shelf life curve' of our system which seems to be the method used in industry to represnet how a TTI works (Labuza,2006).  Secondly I would like to present how system behaves under some dynamic temperature conditions, representing the potential breakdowns in the cold chain as this is the main area in which TTI are useful (Tauokis,2006).
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 !!


Now working backwards from this end point I can see what needs to be done:
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.


#Define & verify shelf lifes for meat (for a given packaging & type) under as many temperature scenarios as possible
==Construct Selection==
#Research which of the scenarios in 1 we can re-create in our lab
##Temps above room temp : can leave for days on end
##Temps below room temp :
### 4 degrees can leave for days on end
### Any other temps will can only do for 9-5 (6 hour period)
### May be able to achieve above by shuttling stuff between Kirsten's lab and our own (not a very good method)
#Precict what will happen in each scenario we can re-create eg. at 4 degrees C we may not get a visible signal after 8 days
#Research increasing lifespan of sytem by adding ATP and t-RNA at the start of system life
#Determine optimum level of DNA concentration to use
##This will happen tomorrow for pTET at 37 degrees C ( as this is a temp we are sure to get expression at)
#Determine packaging for system eg. PCR tube so that evaporation won't be a problem
#Calibration curve for DsRed Express so that we can give meaning to our fluoresence levels


Rough Timetable:
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.


Thrusday 27th Setpember :
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.
*Determine time taken to go off at 37 degrees C to see if we can use conc expt for something else
*Check we have enough maxiprep DNA for tomorrow
*Check we have ordered another batch of cell extract for next week


Friday 28th September : Determine Temperature Scenarios for next week
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.
 
Monday 1st October - Friday 5th October :
*pTET-Mut3B temperature scenario tests
*DsRed Calibration curve & Degradation tests
 
Sunday 7th October : Preliminary Presentation (20 slides in total) and Wiki submission deadline
 
Monday 8th October - Friday 14th October : pTET-DsRED temperature scenario tests
                                         
Wednesday 10th October : Preliminary Write Up (presentation & wiki) Review : decide what left to be done

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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