Imperial/Cell by Date/Design

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  <ul>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction" title=""><span>Introduction</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Specification" title=""><span>Specifications</span></a></li>
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    <li><a class="current" href="https://2007.igem.org/Imperial/Cell_by_Date/Design" title=""><span>Design</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Modelling" title=""><span>Modelling</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Implementation" title=""><span>Implementation</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Testing" title=""><span>Testing</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li>
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__NOTOC__
__NOTOC__
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= Cell by Date : Design=
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= Cell by Date : Overview of Design=
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== Design Overview ==
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[[Image:CellByDateDesignWiki.png]]
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[[Image:IC07 design CBD-bb1.png|center||600px]]
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<br>
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{|
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{|border="1"
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|-
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!Property:
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!Value
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!Design Solution
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!System Level
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|-
|-
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|width="100px"|'''Property'''
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|width="300px"|'''Value'''
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|width="300px"|'''Design Solution'''
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|width="100px"|'''System Level'''
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|- style="background:#eeffee"
|Health Regulations
|Health Regulations
|System Must not be living replicating bacteria
|System Must not be living replicating bacteria
|Use a Cell Free System e.g. Promega's S30 Cell Extract
|Use a Cell Free System e.g. Promega's S30 Cell Extract
|Chassis
|Chassis
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|-
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|- style="background:#eeffee"
|Lifespan
|Lifespan
|System must have a shelf life of 7 days
|System must have a shelf life of 7 days
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|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
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|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 system can surive for 7 days
|Chassis
|Chassis
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|-
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|- style="background:#eeeeff"
|Inputs
|Inputs
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|Isothermal Conditions between 0 & 40 C
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|Isothermal Conditions between 0 & 40 &deg;C
|Exploit Thermal Dependance of rates of expression
|Exploit Thermal Dependance of rates of expression
|Construct
|Construct
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|-
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|- style="background:#eeeeff"
|
|
|Dynamic conditions eg. steps & ramps  
|Dynamic conditions eg. steps & ramps  
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|                                                "
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|style="text-align:center;"|                                                "
|Construct
|Construct
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|-
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|- style="background:#eeeeff"
|Outputs
|Outputs
|System should give a visual signal <br> when beef is off  
|System should give a visual signal <br> when beef is off  
|Couple constituitive promoter to a Fluoresent Protein eg. RFP
|Couple constituitive promoter to a Fluoresent Protein eg. RFP
|Construct
|Construct
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|-
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|- style="background:#eeeeff"
|Activation Energy
|Activation Energy
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|System Needs to have an activation Energy 30 +/- kJ/mol
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|System Needs to have an activation Energy 30 +/- kJ mol<sup>-1</sup>
|To be Determined - this is hard to design for
|To be Determined - this is hard to design for
|Construct
|Construct
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|-
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|- style="background:#eeeeff"
|Response Time
|Response Time
|System needs to have a response time under 1 hour
|System needs to have a response time under 1 hour
|To be Determined - this is hard to design for
|To be Determined - this is hard to design for
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|
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| Both
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|}
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==Chassis Selection==
==Chassis Selection==
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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 !!
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[[Image:IC07_CFS_components.png|thumb|left|340px|Commercial S30 ''E. coli'' Cell Extract in bulk solution + packaging to last 7 days]]
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We have chosen to use the commerciall available S30 ''E. coli'' cell extract made by Promega.  After having looking into a variety of different [[Imperial/Cell-Free/Whatis|cell-free chassis]], we feel that this chassis best suits our needs.  In particular this chassis allows us to meet our base requirement of complying 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.
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.
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==Construct Selection==
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==DNA Constructs==
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'''Concept 1'''<br />
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[[Image:IC07 design CBDcon1.png|center||550px]]
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'''Concept 2'''<br />
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[[Image:IC07 design CBDcon2.png|center||550px]]
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<!--
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{|border=1 width=90% align="center"
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|-  style="background:#eeeeff"
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|width="400px"|'''Construct Design'''
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|width="300px"|'''Availability'''
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|-
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|width="30%"|<center>[[Image:Imperial CBD Design CBD1.png ]]</center>
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|width="--"|This part can be found as [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] in the registry.
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|-
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|<center>[[Image:Imperial CBD Design CBD2.1.png]]</center>
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|width="--"|This part can be found as [http://partsregistry.org/Part:BBa_E7104 BBa_E7104] in the Registry, albeit a different RBS and terminator. May require modifications.
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|}
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-->
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In previous iGEM projects, Temperature as an input has been explored through cold shock and heat shock promoters.  These promoters essentially only operatre for a limited range of temperatures.
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In making a Time Temperature Integrator we would like a promoter that works over a wide range of 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.<sup>[[#References |3]]</sup>
 +
 
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In terms of Activation 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.
 +
 
-
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.
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<center> [https://2007.igem.org/Imperial/Cell_by_Date/Specification << Specifications ] | Design | [https://2007.igem.org/Imperial/Cell_by_Date/Modelling Modelling >>]</center>
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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.
+
== References ==
 +
# [http://jb.asm.org/cgi/content/full/180/17/4704?view=long&pmid=9721314 Anne Farewell and Frederick C. Neidhardt. Effect of Temperature on In Vivo Protein Synthetic Capacity in Escherichia coli. J Bacteriol. 1998 September; 180(17): 4704–4710. ]
 +
# [http://www.clemson.edu/clemsonworld/winter2002/6.htm Kent 2002 : Brief about Vitsab TTI]
 +
# [http://jb.asm.org/cgi/reprint/151/2/879?view=long&pmid=6178724 J Ryals, R Little, and H Bremer. Temperature Dependence of RNA Synthesis Parameters in Escherichia coli. J Bacteriol. 1982 August; 151(2): 879–887. ]

Latest revision as of 02:34, 27 October 2007



Cell by Date : Design

Design Overview

IC07 design CBD-bb1.png


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-1 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 Both


Chassis Selection

Commercial S30 E. coli Cell Extract in bulk solution + packaging to last 7 days

We have chosen to use the commerciall available S30 E. coli cell extract made by Promega. After having looking into a variety of different cell-free chassis, we feel that this chassis best suits our needs. In particular this chassis allows us to meet our base requirement of complying 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.



DNA Constructs

Concept 1

IC07 design CBDcon1.png



Concept 2

IC07 design CBDcon2.png

In previous iGEM projects, Temperature as an input has been explored through cold shock and heat shock promoters. These promoters essentially only operatre for a limited range of temperatures.

In making a Time Temperature Integrator we would like a promoter that works over a wide range of 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.3

In terms of Activation 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.


<< Specifications | Design | Modelling >>

References

  1. [http://jb.asm.org/cgi/content/full/180/17/4704?view=long&pmid=9721314 Anne Farewell and Frederick C. Neidhardt. Effect of Temperature on In Vivo Protein Synthetic Capacity in Escherichia coli. J Bacteriol. 1998 September; 180(17): 4704–4710. ]
  2. [http://www.clemson.edu/clemsonworld/winter2002/6.htm Kent 2002 : Brief about Vitsab TTI]
  3. [http://jb.asm.org/cgi/reprint/151/2/879?view=long&pmid=6178724 J Ryals, R Little, and H Bremer. Temperature Dependence of RNA Synthesis Parameters in Escherichia coli. J Bacteriol. 1982 August; 151(2): 879–887. ]
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