Imperial/Infector Detector/Design

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  <ul>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li>
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    <li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li>
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    <li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li>
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__NOTOC__
= Infector Detector: Design =
= Infector Detector: Design =
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== Design Overview ==
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<center>[[Image:IC07 design ID-bb1.png]] </center>
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<center>[[Image:InfectorDetectorDesignWiki.png]] </center>
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{|border="1"
{|border="1"
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|-
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|width="100px"|'''Property'''
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|width="300px"|'''Specification'''
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|width="300px"|'''Value'''
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|width="420px"|'''Design Solution'''
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|width="300px"|'''Design Solution'''
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|width="100px"|'''System Level'''
|width="100px"|'''System Level'''
|- style="background:#eeffee"
|- style="background:#eeffee"
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|Health Regulations
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|'''Health & Safety''' - System must not contain living cells, or cause any harm to patient.
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|System Must not be living replicating bacteria
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|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
|- style="background:#eeffee"
|- style="background:#eeffee"
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|Lifespan
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|'''Shelf-life''' - System must have a shelf life of 7 days.
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|System must have a shelf life of 7 days
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|Use a Cell Free System that can be packaged and stored in a -80&deg;C freezer 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
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|Chassis
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|- style="background:#eeffee"
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|'''Packaging and Application''' - System must be applied as a cream or spray.
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|Package the system in liquid form.
|Chassis
|Chassis
|- style="background:#eeeeff"
|- style="background:#eeeeff"
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|Inputs
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|'''Inputs''' - System must be sensitive to AHL concentration between 5-50nM.
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|System must be sensitive to AHL concentration between 5-50nM
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|Sensitive AHL receiver to detect low AHL levels - Part no. F2620.
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|Sensitive AHL receiver to detect low AHL levels
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|Construct
|Construct
|- style="background:#eeeeff"
|- style="background:#eeeeff"
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|Outputs  
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|'''Outputs''' - System must give a visual signal if AHL is present.
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|System must give a visual signal if bacteria is present
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|Couple AHL to a reporter system expressing fluoresent protein eg. RFP
|Couple AHL to a reporter system expressing fluoresent protein eg. RFP
|Construct
|Construct
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|- style="background:#eeeeff"
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|- style="background:#ffeeee"
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|Operating Conditions  
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|'''Operating Conditions''' - System must operate within temperature 20&deg;-30&deg;C.
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|System must operate within temperature 20-30°C
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|Gene expression systems and protein must be thermostable. Characterise a Cell Free System at these temperatures.
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|Gene expression systems and protein must be thermostable
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|Construct & Chassis
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|Construct
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|- style="background:#ffeeee"
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|- style="background:#eeeeff"
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|'''Response Time''' - System needs to have a response time under 3 hours.
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|
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|To be Determined - this is hard to design for. Protease Inhibitor of Cell Extract should ensure degradation of Visual Reporter is minimal.
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|System must operate within pH range of 6-8
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|Construct & Chassis
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|Gene expression systems and protein must be pH resistant
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|Construct
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|- style="background:#eeeeff"
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|Response Time
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|System needs to have a response time under 1 hour
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|To be Determined - this is hard to design for
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|Both
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==Chassis Selection==
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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 commercially available S30 ''E. coli'' cell extract made by Promega.  After looking into a variety of different [[Imperial/Cell-Free/Whatis|cell-free systems]], we decided that this chassis is most suitable.  In particular, it allows us to comply with the Health and Safety regulations of the field we are working in, as we do not want replicative bacteria that could potentially be pathogenic to come in contact with urinary catheters.
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== DNA Constructs ==
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In addition to complying with health regulations, the S30 cell extract is commercially available, meaning that it has been shown to work. <sup>[[#References|1]]</sup> This allows our focus to be on tuning the chassis to suit our needs, rather than trying to make the chassis work in the first place.
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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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While
 
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== DNA Constructs ==
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'''Concept 1'''<br /> <center>[[Image:IC07_design_ID.png|550px]]</center>
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Our aim was to create a system that can quickly detect a small (5nM) concentration of AHL in solution. We needed a well characterized detector system sensitive to 3OC6HSL which we will attach to a standard GFP reporter to determine its response to GFP. The first construct is appealing as the AHL receiver in front is well-documented as part [http://partsregistry.org/Part:BBa_F2620 BBa_F2620], thus giving more assurance in the reproducible nature of our results.
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'''Concept 2'''<br /> <center>[[Image:IC07_design_ID-con2.png|550px]]</center>
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However, the weakness of concept 1 lies in the fact that it does not respond uniformly to constant AHL concentration since the activator protein LuxR is produced by a promoter and not maintained at a constant level. This makes it hard to relate the output of the system to the AHL input. However, if purified LuxR could be added to the system instead, the construct could be simplified by removing the constitutive LuxR production. This would give us more control of the input, and therefore better reproducibility.
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==Chassis Selection==
 
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{| style="width:800px;" cellpadding="2" cellspacing="0" align="center"
 
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|- style="background:#ffffff; text-align:left;" color:white;" align="center"
 
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|| [[Image:Icgems-bulksol.png|180px]]
 
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||
 
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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, as we do not want replicative bacteria that could potentially be pathogenic to come in contact with urinary catheters.
 
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|- style="background:#ffffff; text-align:left;" color:white;" align="center"
 
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|style="font-size:0.85em;"| Commercial S30 ''E. coli'' Cell Extract in bulk solution + packaging to last 7 days
 
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||
 
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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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|}
 
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<center>  [https://2007.igem.org/Imperial/Infector_Detector/Specification << Specification] | Design | [https://2007.igem.org/Imperial/Infector_Detector/Modelling Modelling >>]
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</center>
== References ==
== References ==
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 +
# [https://promega.de/tbs/tb092/tb092.pdf Promega S30 Cell Extract documentation]

Latest revision as of 02:34, 27 October 2007



Infector Detector: Design

IC07 design ID-bb1.png


Specification Design Solution System Level
Health & Safety - System must not contain living cells, or cause any harm to patient. Use a Cell Free System e.g. Promega's S30 Cell Extract Chassis
Shelf-life - System must have a shelf life of 7 days. Use a Cell Free System that can be packaged and stored in a -80°C freezer for 7 days. Chassis
Packaging and Application - System must be applied as a cream or spray. Package the system in liquid form. Chassis
Inputs - System must be sensitive to AHL concentration between 5-50nM. Sensitive AHL receiver to detect low AHL levels - Part no. F2620. Construct
Outputs - System must give a visual signal if AHL is present. Couple AHL to a reporter system expressing fluoresent protein eg. RFP Construct
Operating Conditions - System must operate within temperature 20°-30°C. Gene expression systems and protein must be thermostable. Characterise a Cell Free System at these temperatures. Construct & Chassis
Response Time - System needs to have a response time under 3 hours. To be Determined - this is hard to design for. Protease Inhibitor of Cell Extract should ensure degradation of Visual Reporter is minimal. Construct & Chassis


Chassis Selection

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

We have chosen to use the commercially available S30 E. coli cell extract made by Promega. After looking into a variety of different cell-free systems, we decided that this chassis is most suitable. In particular, it allows us to comply with the Health and Safety regulations of the field we are working in, as we do not want replicative bacteria that could potentially be pathogenic to come in contact with urinary catheters.

In addition to complying with health regulations, the S30 cell extract is commercially available, meaning that it has been shown to work. 1 This allows our focus to be on tuning the chassis to suit our needs, rather than trying to make the chassis work in the first place.



DNA Constructs

Concept 1
IC07 design ID.png

Our aim was to create a system that can quickly detect a small (5nM) concentration of AHL in solution. We needed a well characterized detector system sensitive to 3OC6HSL which we will attach to a standard GFP reporter to determine its response to GFP. The first construct is appealing as the AHL receiver in front is well-documented as part [http://partsregistry.org/Part:BBa_F2620 BBa_F2620], thus giving more assurance in the reproducible nature of our results.


Concept 2
IC07 design ID-con2.png


However, the weakness of concept 1 lies in the fact that it does not respond uniformly to constant AHL concentration since the activator protein LuxR is produced by a promoter and not maintained at a constant level. This makes it hard to relate the output of the system to the AHL input. However, if purified LuxR could be added to the system instead, the construct could be simplified by removing the constitutive LuxR production. This would give us more control of the input, and therefore better reproducibility.



<< Specification | Design | Modelling >>

References

  1. Promega S30 Cell Extract documentation
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