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

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m (Infector Detector: Design)
Current revision (08:16, 11 September 2007) (view source)
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==Overall Design - Construct 1==
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==Overall Design==
===Possible Parts===
===Possible Parts===
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Peak secretion: 400 GFP molecules cfu<sup>-1</sup> s<sup>-1</sup><br>
Peak secretion: 400 GFP molecules cfu<sup>-1</sup> s<sup>-1</sup><br>
Peak response time: 27 minutes<br>
Peak response time: 27 minutes<br>
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Part [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 BBa_T9002] already has GFP attached
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[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 BBa_T9002] ptet-LuxR-pLux-GFP<br>
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With GFP attached<br>
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[http://parts.mit.edu/registry/index.php/Part:BBa_J37032 BBa_J37032] pLux-GFP<br>
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The same part, but without constitutive LuxR production
====GFP Reporter====
====GFP Reporter====
[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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<s>LacZ for &beta;-galactosidase assay (yet to be discovered...)</s><br>
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<s>LacZ for &beta;-galactosidase assay (LacZ is too big...)</s><br>
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Or possibly DsRed Express!
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Or possibly DsRed Express - this will be ordered from clontech!
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==Stage 1: Detector for AHL==
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==Construct 1: Detector for AHL==
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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 3OC<sub>6</sub>HSL which we will attach to a standard GFP reporter to determine its response to GFP.<br>&nbsp;<br>
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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 3OC<sub>6</sub>HSL which we will attach to a standard GFP reporter to determine its response to GFP.<br>
<br>[[Image:IC2007_BF_stage1_construct.png]]<br clear="all">&nbsp;<br>
<br>[[Image:IC2007_BF_stage1_construct.png]]<br clear="all">&nbsp;<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>
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>
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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].
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].
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==Stage 2: Detector for bacterial secreted AHL==
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==Component: Detector for bacterial secreted AHL==
To more accurately simulate a bacterial biofilm, we decided to create bacteria that would constitutively secrete AHL.<br>
To more accurately simulate a bacterial biofilm, we decided to create bacteria that would constitutively secrete AHL.<br>
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<br>[[Image:IC2007_BF_stage2_construct_sender.png]]<br clear="all>&nbsp;<br>
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<br>[[Image:IC2007_BF_stage2_construct_sender.png]]<br clear="all>
 +
We might need to clone this part in if the biofilm ''E. coli'' strain doesn't produce AHL by itself...
 +
==Component: Amplifier for reporter production==
Our detector plasmid would incorporate the Hrp system as an amplifier, to amplify a reporter gene yet to be decided.<br>
Our detector plasmid would incorporate the Hrp system as an amplifier, to amplify a reporter gene yet to be decided.<br>
<br>[[Image:IC2007_BF_stage2_construct_receiver.png]]<br clear="all>&nbsp;<br>
<br>[[Image:IC2007_BF_stage2_construct_receiver.png]]<br clear="all>&nbsp;<br>
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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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''It has now been shown that the amplifier system will most likely not work. This part of the project has been cancelled.''
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http://www.openwetware.org/wiki/Image:IC2007_BF_stage1_construct2.png
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==Construct 2: Detector for AHL==
 +
From our models, it has been shown that the construct 1 does not respond uniformly, 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. We decided to add purified LuxR to the system instead, and simplify the construct by removing the constitutive LuxR production.<br>
 +
<br>[[Image:IC2007_BF_stage1_construct2.png]]

Current revision

Infector Detector: Design



Overall Design

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
BBa_T9002 ptet-LuxR-pLux-GFP
With GFP attached
BBa_J37032 pLux-GFP
The same part, but without constitutive LuxR production

GFP Reporter

BBa_EI1354 GFP
Standard GFP, excited/emitted at 501/511 nm, with RBS and stop codons.
LacZ for β-galactosidase assay (LacZ is too big...)
Or possibly DsRed Express - this will be ordered from clontech!

Construct 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].

Component: 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
We might need to clone this part in if the biofilm E. coli strain doesn't produce AHL by itself...

Component: Amplifier for reporter production

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.

It has now been shown that the amplifier system will most likely not work. This part of the project has been cancelled.

Construct 2: Detector for AHL

From our models, it has been shown that the construct 1 does not respond uniformly, 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. We decided to add purified LuxR to the system instead, and simplify the construct by removing the constitutive LuxR production.

Image:IC2007_BF_stage1_construct2.png

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