Team:Cambridge/Project/Amplification/Characterisation

From 2009.igem.org

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In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC.  These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects.  For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at four major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.
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In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC.  These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects.  For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.
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[[Image:characterization1.jpg]]
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[[Image:characterization2.jpg]]
== Characterisation ==
== Characterisation ==

Revision as of 21:09, 21 October 2009


The Sensitivity Tuner

Introduction

The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (), as described below:

Amplifier07.jpg

We re-designed these constructs to be PoPS converters as follows...

Thresholddevice3.jpg

...and generated our own set of Sensitivity Tuners:

P2 ogr activator PSP3 pag activator phiR73 delta activator
PF promoter
PO promoter
PP promoter
Psid promoter
PLL promoter

In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with , the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.

Characterization2.jpg

Characterisation

We moved all 15 activator constructs onto pSB3K3, a low copy plasmid. The standard promoter for 1 RPU, J69591, is also on pSB3K3 and has a GFP reporter, so we can make meaningful comparisons on the plate reader.

Maximum Rates against Arabinose Concentrations

80

Cambridge maxrates1.jpg

81

Cambridge maxrates2.jpg

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Cambridge maxrates3.jpg

84

Cambridge maxrates4.jpg

85

Cambridge maxrates5.jpg

90

Cambridge maxrates6.jpg

92

Cambridge maxrates7.jpg

94

Cambridge maxrates8.jpg

95

Cambridge maxrates9.jpg


Cambridge Sponsor Logo1.pngCambridge Sponsor Logo2.pngCambridge Sponsor Logo3.pngCambridge Sponsor Logo4.pngCambridge Sponsor Logo5.pngCambridge Sponsor Logo8.pngCambridge Sponsor Logo6.pngCambridge Sponsor Logo7.pngCambridge Sponsor Logo9.pngCambridge Sponsor Logo10.pngCambridge Sponsor Logo11.pngCambridge Sponsor Logo12.pngCambridge Sponsor Logo14.pngCambridge Sponsor Logo13.pngCambridge Sponsor Logo15.pngCambridge Sponsor Logo16.pngCambridge Sponsor Logo17.pngCambridge Sponsor Logo18.pngCambridge Sponsor Logo19.pngBmglab.jpg