IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Design

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(Stage 2: Detector for bacterial secreted AHL)
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==Overall Design==
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==Overall Design - Construct 1==
===Possible Parts===
===Possible Parts===
====Signaling System====
====Signaling System====
[http://parts.mit.edu/registry/index.php/Part:BBa_F1610 BBa_F1610] RBS-luxI<br>
[http://parts.mit.edu/registry/index.php/Part:BBa_F1610 BBa_F1610] RBS-luxI<br>
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No promoter - we need to put one on it!
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No promoter - we need to put one on it!<br>
 +
[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 BBa_J23039] ptet-luxI<br>
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A better part has been found! This part will most likely be used.
====Promoter for Signaling System====
====Promoter for Signaling System====
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ptet, if uncontrolled
ptet, if uncontrolled
<br>
<br>
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pLac, if constitutive secretion<br>
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<s>pLac, if constitutive secretion<br>
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pBAD, if controlled secretion
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pBAD, if controlled secretion</s>
====Detection System====
====Detection System====
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[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 BBa_EI1354] GFP<br>
[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 BBa_EI1354] GFP<br>
Standard GFP, excited/emitted at 501/511 nm, with RBS and stop codons.<br>
Standard GFP, excited/emitted at 501/511 nm, with RBS and stop codons.<br>
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LacZ for &beta;-galactosidase assay (yet to be discovered...)
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<s>LacZ for &beta;-galactosidase assay (yet to be discovered...)</s><br>
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Or possibly DsRed Express!
==Stage 1: Detector for AHL==
==Stage 1: Detector for AHL==
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We would later test the promoter on a different reporter. The reporter that is chosen for the final comstruct needs to have a long half-life, be stable, be visible to the naked eye and should not have a response time longer than 3 hours. <br>
We would later test the promoter on a different reporter. The reporter that is chosen for the final comstruct needs to have a long half-life, be stable, be visible to the naked eye and should not have a response time longer than 3 hours. <br>
<br>[[Image:IC2007_BF_stage1a_construct.png‎]]<br clear="all">
<br>[[Image:IC2007_BF_stage1a_construct.png‎]]<br clear="all">
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It has been decided that DsRed Express will be the final reporter, since it has two important properties. A very long half-life (several days), and is visible to the naked eye. [The second property might not be true for small concentrations of DsRed].
==Stage 2: Detector for bacterial secreted AHL==
==Stage 2: Detector for bacterial secreted AHL==
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The characterisation of the '''[[IGEM:IMPERIAL/2007/Projects/Hrp System|Hrp System]]''' will be carried out along with this project, and thus will only be incorporated into the infector detector once the system has been fully characterised.
The characterisation of the '''[[IGEM:IMPERIAL/2007/Projects/Hrp System|Hrp System]]''' will be carried out along with this project, and thus will only be incorporated into the infector detector once the system has been fully characterised.
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==Overall Design - Construct 2==
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 +
http://www.openwetware.org/wiki/Image:IC2007_BF_stage1_construct2.png

Revision as of 07:02, 11 September 2007

Infector Detector: Design



Overall Design - Construct 1

Possible Parts

Signaling System

BBa_F1610 RBS-luxI
No promoter - we need to put one on it!
BBa_J23039 ptet-luxI
A better part has been found! This part will most likely be used.

Promoter for Signaling System

BBa_R0040 ptet, if uncontrolled
pLac, if constitutive secretion
pBAD, if controlled secretion

Detection System

BBa_F2620 ptet-LuxR-pLux
Detection range: 1-1000 nM, highest sensitivity from 1-10 nM
Peak secretion: 400 GFP molecules cfu-1 s-1
Peak response time: 27 minutes
Part BBa_T9002 already has GFP attached

GFP Reporter

BBa_EI1354 GFP
Standard GFP, excited/emitted at 501/511 nm, with RBS and stop codons.
LacZ for β-galactosidase assay (yet to be discovered...)
Or possibly DsRed Express!

Stage 1: Detector for AHL

Our first aim is to create a system that can accurately detect the concentration of AHL in solution. We found a well characterized detector system sensitive to 3OC6HSL which we will attach to a standard GFP reporter to determine its response to GFP.
 

Image:IC2007_BF_stage1_construct.png
 
We would later test the promoter on a different reporter. The reporter that is chosen for the final comstruct needs to have a long half-life, be stable, be visible to the naked eye and should not have a response time longer than 3 hours.

Image:IC2007_BF_stage1a_construct.png‎

It has been decided that DsRed Express will be the final reporter, since it has two important properties. A very long half-life (several days), and is visible to the naked eye. [The second property might not be true for small concentrations of DsRed].

Stage 2: Detector for bacterial secreted AHL

To more accurately simulate a bacterial biofilm, we decided to create bacteria that would constitutively secrete AHL.

Image:IC2007_BF_stage2_construct_sender.png
 

Our detector plasmid would incorporate the Hrp system as an amplifier, to amplify a reporter gene yet to be decided.

Image:IC2007_BF_stage2_construct_receiver.png
 

The characterisation of the Hrp System will be carried out along with this project, and thus will only be incorporated into the infector detector once the system has been fully characterised.

Overall Design - Construct 2

http://www.openwetware.org/wiki/Image:IC2007_BF_stage1_construct2.png

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