Team:Michigan/Results

From 2012.igem.org

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Introduction
Introduction
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In order to test the effectiveness of the recombinases used in our system, we developed two assays -- an “asymmetrical digest” reporter and a fluorescence reporter.  The former is used to directly test the inversion of the target DNA region, while the latter is used to demonstrate inversion induced protein expression.
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In order to test the effectiveness of the recombinases used in our system, we developed two assays -- an “asymmetrical digest” reporter and a fluorescence reporter. The former is used to directly test the inversion of the target DNA region, while the latter is used to demonstrate inversion induced protein expression.  
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The two asymmetrical digest reporters in their unflipped state (invertible by FimE) are shown in figures 2 and 3. These reporters rely on an off-center AgeI restriction site which will yield fragments of varying size depending on the orientation of the inversion region. Table 1 shows the expected sizes of the fragments in each orientation. The differences between the reporters are discussed below.  
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The Fluorescent Reporters
The Fluorescent Reporters
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The fluorescent reporters, show in in figures 3 and 4, have a constitutive tetR promoter within the inversion site, allowing for the expression of GFP in the flipped orientation.  
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The two asymmetrical digest reporters in their unflipped state (invertible by FimE) are shown in figures 2 and 3. These reporters rely on an off-center AgeI restriction site which will yield fragments of varying size depending on the orientation of the inversion region. Table 1 shows the expected sizes of the fragments in each orientation. The differences between the reporters are discussed below.  
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Revision as of 00:16, 4 October 2012

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Introduction

In order to test the effectiveness of the recombinases used in our system, we developed two assays -- an “asymmetrical digest” reporter and a fluorescence reporter. The former is used to directly test the inversion of the target DNA region, while the latter is used to demonstrate inversion induced protein expression.

The Asymmetrical Digest Reporters

The two asymmetrical digest reporters in their unflipped state (invertible by FimE) are shown in figures 2 and 3. These reporters rely on an off-center AgeI restriction site which will yield fragments of varying size depending on the orientation of the inversion region. Table 1 shows the expected sizes of the fragments in each orientation. The differences between the reporters are discussed below.

The Fluorescent Reporters

The fluorescent reporters, show in in figures 3 and 4, have a constitutive tetR promoter within the inversion site, allowing for the expression of GFP in the flipped orientation.

Figure 1. A diagram of the asymmetrical reporter K880001




Figure 2. A diagram of the asymmetrical reporter K880003

Results

Negative Fluorescent Reporter (BBa_K137058) Data

Negative Asymmetrically Digestible Reporter (BBa_K880003) Data

Observation of IRR and IRL Orientation:

Modified Asymmetrically Digestible Reporter (BBa_K880001) Data:

Negative Modified Fluorescent Reporter (BBa_K880002) Data:

Observation of HbiF induced fimbriation in DH5alpha

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