Team:Michigan/Results
From 2012.igem.org
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Negative Asymmetrically Digestible Reporter (BBa_K880003) Data | Negative Asymmetrically Digestible Reporter (BBa_K880003) Data | ||
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+ | An asymmetrical digest reporter (K880003) in the vector pSB3C5 based on the inversion region found in K137058 was co-transformed in 10-beta <i>E. coli</i> (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2. Colonies as well as broth subcultures of co-transformants were analyzed for inversion using the asymmetrical digest assay (hyperlink to protocol) . The asymmetrical digest reporter and <i>fimE</i> are present within the co-transformants; however, the asymmetrical digest reporter remains in the uninverted state (Fig 5, lane 3). | ||
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Revision as of 00:17, 4 October 2012
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.