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Team:Penn/LightActivatedLysis
From 2012.igem.org
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| - | <b><div class="name" align="center">Verification of | + | <b><div class="name" align="center">Verification of Expression of Cytolysin A (ClyA)</div></b><br /> |
<div class="fig"><div align="center"><img src="http://partsregistry.org/wiki/images/3/3a/Gel_pic_pdawn.png"><br> | <div class="fig"><div align="center"><img src="http://partsregistry.org/wiki/images/3/3a/Gel_pic_pdawn.png"><br> | ||
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Revision as of 13:14, 26 October 2012
We then wanted to prove that our pDawn-ClyA construct was able to lyse mammalian cells in a light-dependent manner. To assess this, we plated BL21 bacteria transformed with pDawn-ClyA or pDawn-mCherry on Columbia Agar plates supplemented with 5% Sheep Blood (BD). These plates are used to qualitatively detect hemolytic activity in bacteria by visually confirming lysis through a color change in the media as the blood cells are lysed. After plating the bacteria, cultures were grown in non-inducing conditions at 37C until visible colonies were present (~12 hours). Plates were then grown at 25C under either inducing or non-inducing conditions for 24 hours and imaged. These results are visible in Figure 4.
pDawn-mCherry Dark
pDawn-mCherry Light
pDawn-His-ClyA Dark
pDawn-His-ClyA Light
Figure 5
Figure 5: Colony Spatial Control.
Figure 6
Figure 6: Penn iGEM spatial control
Figure 7
Figure 7: The production of clyA-his in BL21 in both bacteral lysate and culture medium
In order to further develop our system for future in vivo therapeutic applications, we transformed Nissle 1917 with pDawn-mCherry to see if we could implement our system into a non-pathogenic strain of E. coli. We repeated our initial experiments and achieved light-dependent gene expression in Nissle 1917 for the first time ever. We are now hoping to clone in our pDawn-ClyA construct to show that Nissle 1917 is capable of light-dependent lysis of mammalian cells. Stay tuned!
Figure 8
