Team:Linkoping Sweden/Results

From 2014.igem.org

(Difference between revisions)
Line 45: Line 45:
<h2 class="clickable ">Biobricks</h2>
<h2 class="clickable ">Biobricks</h2>
<div id="biobricksexp" class="expandable">
<div id="biobricksexp" class="expandable">
-
<p>These discussed combinations resulted in 4 different Biobricks which are the two combinations of RFP linked to either epitope 2 (Fig. 1) or all 5 epitopes (Fig. 2) as well as the two combinations of MCherry with either epitope 2 or all 5 epitopes.  Protein expressed from the Biobrick including epitope 2 linked to RFP/MCherry will thus be used in combination with the monoclonal antibodies which are specific for epitope 2 as mentioned previously. The 5 different epitopes linked to RFP/MCherry will be used in combination with the polyclonal antibodies. Worth mentioning as well is that all the 4 biobricks contain a sequence for Promotor + RBS as well as a sequence for His-TEV. Furthermore, we designed a fifth Biobrick as well including a sequence for Promotor + RBS and a sequence for His-TEV (Fig. 3).</p>
+
<p>These discussed combinations resulted in 4 different Biobricks which are the two combinations of RFP linked to either epitope 2 (Fig. 1) or all 5 epitopes (Fig. 2) as well as the two combinations of MCherry with either epitope 2 or all 5 epitopes.  Protein expressed from the Biobrick including epitope 2 linked to RFP/MCherry will thus be used in combination with the monoclonal antibodies which are specific for epitope 2 as mentioned previously. The 5 different epitopes linked to RFP/MCherry will be used in combination with the polyclonal antibodies. Worth mentioning as well is that all the 4 biobricks contain a sequence for Promotor + RBS as well as a sequence for His-TEV. Furthermore, we designed an additional fifth Biobrick including a sequence for Promotor + RBS and a sequence for His-TEV (Fig. 3).</p>
<div class="image-box" style="width:1000px">
<div class="image-box" style="width:1000px">

Revision as of 11:03, 17 October 2014

Our vision is to create a Biobrick which includes the sequence of the Ara h1 protein linked to a red fluorescent protein. We want to accomplish this so that we in turn can express the protein and thus provide an interaction of this protein complex with the antibodies. To ensure that the epitope-red fluorescent protein complex will bind to the Ara h1 specific IgG antibodies several ideas for Biobrick-design was brought to mind. Since we use both monoclonal antibodies specific for epitope 2 on Ara h1 and polyclonal antibodies specific for several epitopes on Ara h1 we decided to create a Biobrick consisting of epitope 2 of Ara h1 linked to a red fluorescent protein as well as a biobrick consisting of five wisely chosen epitopes (epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17) from Ara h1 linked to a red fluorescent protein. However, since there are two different mutants of red fluorescent protein (called RFP and MCherry respectively) we decided to create two setups of every Biobrick combination to ensure that the best possible detection by FRET is attained, as RFP and MCherry differ slightly in their excitation and emission wavelengths. The main idea is to practically test this FRET effect in both epitope-RFP and epitope-MCherry combinations by fluorescence. The data from these tests will show us which mutant is best suited to use and thus prove that our theory works.

Linköping University
581 83 Linköping, Sweden
liuigemgroup@gmail.com
Copyright (c) 2014 igem.org. All rights reserved.