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Team:Cambridge/Project/Amplification
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Revision as of 07:51, 12 August 2009
Categories :
Project :
-
Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
The Adaptor
Introduction
Cambridge iGEM 2007
The Cambridge 2007 iGEM team developed a PoPS amplifier system using phage activators and promoters. The system works by using a PoPS input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a Pops output.
In order to quantify the ratio between PoPS in and PoPS out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as PoPS reporters and 15 total combinations of different activators and reporters.
They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction.
Further work - Cambridge iGEM 2009
We hope more fully characterize the activator constructs. First, we hope to characterize these activators on low copy plasmids as promoters compared to the Relative Promoter Unit (RPU). Secondly, we will investigate the activators' sensitivity to arabinose.
Recreating Previous Work
We began by recreating the 2007 team's data with some select amplifier constructs. We have the advantage over the 2007 team in that we have a better plate reader that is able to take OD600 absorbance readings at the same time as taking RFP and GFP absorbance readings. For our transformations, we used the E. coli host strain BW27783. This host strain constitutively expresses arabinose transporters and is unable to metabolize arabinose, making it an ideal host for arabinose titration experiments.
Characterization
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.
Results
