Team:UIUC-Illinois/Project/Design

From 2012.igem.org

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<p>In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to match both the PUF-PIN and mPUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. The control YFP reporter construct encodes a binding site that doesnt recognize both wtPUF/mPUF protein.</p>
<p>In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to match both the PUF-PIN and mPUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. The control YFP reporter construct encodes a binding site that doesnt recognize both wtPUF/mPUF protein.</p>

Revision as of 06:10, 26 October 2012

Header

Project Design

PUF Project Design

  • Overview
  • PUF+PIN Fusion
  • YFP Reporter
  • Non-Specific Control
  • Experiments
  • Theoretical Results
  • PUF Experimental Design


    In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by two base pairs.

    Click on the list to the left to read about each of our constructs and why we decided to do them. All expressions were done in vivo with the DH5a strain of E.Coli on pBAD30 or pPROTet.E plasmids.

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