Team:Michigan/Results

Introduction

The Asymmetrical Digest Reporters

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 Fluorescent 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.

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