IGEM:IMPERIAL/2008/New/Cloning Strategy

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{{Imperial/StartPage2}}__NOTOC__
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Authors: Chris
 
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Editors: Wetlab team, wiki team
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{{Imperial/Box2|Cloning Strategy|
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The Imperial iGEM 2008 team faces the task of working with a chassis that has been rarely used - and never characterised - in the competition to date. While the ''B. subtilis'' chassis offers us many advantages, working from the ground up also presents many challenges.}}
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= Cloning Strategy =
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{{Imperial/Box1||
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The cloning strategy for our Biofabricator is complex. In order to build the required constructs for our final product, we need to build, test and characterise intermediary parts and devices that will lead to the final system. The diagram below shows the critical pathway for our cloning strategy with a large number of closely-linked steps.
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<br><br>
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[[Image:Imperial_2008_Critical_Pathway.png|center|700px]]
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<br><br>}}
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The Imperial iGEM 2008 team faces the mammoth task of working with a chassis that has been rarely used in the competition so far. This leaves us with many challenges to overcome, in particular having to work from the ground up.
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{{Imperial/Box1|Constructs|
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A summary of the aims of each phase and constructs to be produced within each phase, is given below:
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This also makes our cloning stratergy highly complicated. In order to building increasingly complicated constructs for our final product, we must first build and test simpler parts and devices.
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====== Phase 1: Testing of Constitutive Promoters ======
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We will test 4 combinations of 2 constitutive promoters and 2 RBSs to characterise them. An antibiotic resistance cassette is placed at the 5' end of the construct, to prevent any readthrough by the native trancriptases from reaching the regulated BioBricks.
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<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase1.png"></html>
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[[Image:Imperial_2008_Critical_Pathway.png|center|800px|]]
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====== Phase 2: Testing of Inducible Promoters ======
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Promoters marked with a 'C' are chemically-inducible and those marked with an 'L' are light-inducible. Since YtvA responds to blue light, fluorescence from GFP may cause positive feedback. We will therefore be using RFP as a quantifiable output to test our light-inducible promoters instead. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression.
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<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png">
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<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2B.png"></html>
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====== Phase 3: Clutch and Biomaterial Characterisation ======
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Testing and characterisation of the clutch (EpsE, produced by the ''epsE'' gene) and biomaterial synthesis (SB - signal peptide and biomaterial) using a chemically-inducible promoter. The promoter is otherwise constitutively repressed by Rep.
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<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3A.png">
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<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3B.png"></html>
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This is the overall cloning strategy for our project. A summary of constructs that should be produced by the end of each phase for testing is shown below:
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====== Phase 4: Device Characterisation ======
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Combination of light induction and EpsE/biomaterial expression. The clutch (EpsE) and biomaterial are now under control of the light-inducible activator sigma B (via YtvA).
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{| border="0" cellpadding="2" style="text-align:left;" width=100%
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<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4A.png">
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|-
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<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4B.png"></html>
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! <big>Phase 1
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|-
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====== Final Construct: System Characterisation ======
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| [[Image:Phase_1.PNG|300px]]
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Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter/RBS pair because in ''B. subtilis'', it has been shown that levels of expression decrease as one moves along an operon.
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<p><html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html></p>
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! <big>Phase 2A
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}}
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! <big>Phase 2B
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{{Imperial/EndPage|Wet_Lab|Protocols}}
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| [[Image:Phase_2-linduced.PNG|300px]]
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| [[Image:Phase_2-light_induced.PNG|300px]]
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|-
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! <big>Phase 3A
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! <big>Phase 3B
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|-
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| [[Image:Phase_2-linduced22.PNG|300px]]
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| [[Image:Phase_2-biomaterials.PNG|300px]]
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! <big>Phase 4A
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! <big>Phase 4B
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|-
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| [[Image:Phase_4-clutch.PNG|300px]]
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| [[Image:Phase_4-clutch.PNG|300px]]
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! <Big>Final Construct
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| [[Image:Imperial_2008_Named_Circuitry.jpg|300px]] 
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Current revision





Cloning Strategy

The Imperial iGEM 2008 team faces the task of working with a chassis that has been rarely used - and never characterised - in the competition to date. While the B. subtilis chassis offers us many advantages, working from the ground up also presents many challenges.


The cloning strategy for our Biofabricator is complex. In order to build the required constructs for our final product, we need to build, test and characterise intermediary parts and devices that will lead to the final system. The diagram below shows the critical pathway for our cloning strategy with a large number of closely-linked steps.




Constructs

A summary of the aims of each phase and constructs to be produced within each phase, is given below:

Phase 1: Testing of Constitutive Promoters

We will test 4 combinations of 2 constitutive promoters and 2 RBSs to characterise them. An antibiotic resistance cassette is placed at the 5' end of the construct, to prevent any readthrough by the native trancriptases from reaching the regulated BioBricks.

Phase 2: Testing of Inducible Promoters

Promoters marked with a 'C' are chemically-inducible and those marked with an 'L' are light-inducible. Since YtvA responds to blue light, fluorescence from GFP may cause positive feedback. We will therefore be using RFP as a quantifiable output to test our light-inducible promoters instead. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression.

Phase 3: Clutch and Biomaterial Characterisation

Testing and characterisation of the clutch (EpsE, produced by the epsE gene) and biomaterial synthesis (SB - signal peptide and biomaterial) using a chemically-inducible promoter. The promoter is otherwise constitutively repressed by Rep.

Phase 4: Device Characterisation

Combination of light induction and EpsE/biomaterial expression. The clutch (EpsE) and biomaterial are now under control of the light-inducible activator sigma B (via YtvA).

Final Construct: System Characterisation

Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter/RBS pair because in B. subtilis, it has been shown that levels of expression decrease as one moves along an operon.



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