Team:Penn/LightActivatedLysis

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<b><div class="name" align="center">pDawn and Nissle 1917</div></b><br />
 
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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!
 
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<div class="fig"><div align="center"><img src="http://2012.igem.org/wiki/images/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="250" height="350"><br>
 
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<b>Figure 8</b></div></div>
 
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Revision as of 23:10, 26 October 2012

Penn 2012 iGEM Wiki

Image Map

Light-Dependent Lysis of Mammalian Cells by Bacteria

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 37°C until visible colonies were present (~12 hours). Plates were then grown at 25°C 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 4
Spatial Cell Lysis


Figure 5
Figure 5: Colony Spatial Control.

Figure 6
Figure 6: Penn iGEM spatial control
Verification of Expression of Cytolysin A (ClyA)



Figure 7
Figure 7: The production of clyA-his in BL21 in both bacteral lysate and culture medium

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