Team:Cambridge/Project/Amplification

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The Threshold Device

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.

Amplifier07.jpg


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 promoters.

Construction07.jpg


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 output 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.

Results: The 2007 team's hopes for future work included investigating a problem they attributed to the toxicity of high levels of activator in the cell. Overnight OD600 readings of cells transformed with their amplifier constructs indicated cell death. However, these OD readings were conducted separately from their RFP and GFP output measurements. The 2009 team gathered data on the plate reader capable of taking OD600 absorbance readings as well as RFP and GFP output readings, no OD600 readings suggested cell death due to toxicity.

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

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