IGEM:IMPERIAL/2008/New/Cloning Strategy

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{{Imperial/StartPage2}}__NOTOC__
{{Imperial/StartPage2}}__NOTOC__
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= '''Cloning Strategy''' =
 
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The Imperial iGEM 2008 team faces the daunting task of working with a chassis that has been rarely used - and never characterised - in the competition so far. While the ''subtilis'' chassis offers us many advantages, working from the ground up like this also presents many challenges.
 
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Our cloning strategy, for instance, is highly complex. In order to building increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading up to the final systems! The diagram below shows the "critical pathway" for our cloning strategy and as you can see, there are a huge number of closely-linked steps..
 
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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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{{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>
[[Image:Imperial_2008_Critical_Pathway.png|center|700px]]
[[Image:Imperial_2008_Critical_Pathway.png|center|700px]]
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<br><br>}}
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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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A summary of the aims of the phases, and constructs that should be produced by the end of each for testing, is given below:
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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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====== Phase 1 ======
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Testing and characterisation of constitutive promoters. We will test 4 combinations of 2 promoters and 2 RBSs to characterise them. Antibiotic cassette is placed first on the construct, so that any readthrough from native transcriptase simply boosts production of antibiotic.
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<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase1.png"></html>
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase1.png"></html>
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====== Phase 2 ======
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====== Phase 2: Testing of Inducible Promoters ======
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Testing and characterisation of inducible promoters; those marked with a 'c' are chemically-inducible and those marked with an 'l' are light-inducible. RFP is used instead of GFP to check output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' gene signifies a repressor for the chemically-inducible promoter to stop leaky expression.
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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.
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png">
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png">
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2B.png"></html>
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2B.png"></html>
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====== Phase 3 ======
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====== Phase 3: Clutch and Biomaterial Characterisation ======
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Testing and characterisation of the clutch (''epsE'') and biomaterial synthesis (SB - signal sequence & biomaterial).
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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.
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3A.png">
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3A.png">
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3B.png"></html>
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3B.png"></html>
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====== Phase 4 ======
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====== Phase 4: Device Characterisation ======
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Combining of light induction and epsE/biomaterial expression, and testing of feasibility.
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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).
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4A.png">
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4A.png">
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4B.png"></html>
<img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4B.png"></html>
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====== Final Construct ======
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====== Final Construct: System Characterisation ======
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Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter because in ''B. subtilis'' it has been shown that levels of expression drop as one moves along an operon.
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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.
<p><html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html></p>
<p><html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html></p>
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}}
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{{Imperial/EndPage|Wet_Lab|Protocols}}

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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