Team:Penn/LightActivatedLysis
From 2012.igem.org
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<b>Figure 6</b><br> | <b>Figure 6</b><br> | ||
Figure 6: Penn iGEM spatial control</div></div></div> | Figure 6: Penn iGEM spatial control</div></div></div> | ||
- | <div class="fig"><div align="center"><img src="http://partsregistry.org/ | + | <div class="fig"><div align="center"><img src="http://partsregistry.org/wiki/images/3/3a/Gel_pic_pdawn.png"><br> |
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<b>Figure 7</b><br> | <b>Figure 7</b><br> |
Revision as of 12:08, 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.
![](https://static.igem.org/mediawiki/2012/4/42/PDawn-mCherry-Dark-Grey-BG.jpg)
pDawn-mCherry Dark
![](https://static.igem.org/mediawiki/2012/c/c0/PDawn-mCherry-Light-Grey-BG.jpg)
pDawn-mCherry Light
![](https://static.igem.org/mediawiki/2012/d/db/PDawn-ClyA-His-Dark-Grey-BG.jpg)
pDawn-His-ClyA Dark
![](https://static.igem.org/mediawiki/2012/3/35/PDawn-ClyA-His-Light-Grey-BG.jpg)
pDawn-His-ClyA Light
![](https://static.igem.org/mediawiki/2012/7/76/Colony-Spatial-Lysis---Grey-BG.jpg )
Figure 5
Figure 5: Colony Spatial Control.
![](https://static.igem.org/mediawiki/2012/1/10/Penn-iGEM-Lysis---Grey-BG.jpg)
Figure 6
Figure 6: Penn iGEM spatial control
![](http://partsregistry.org/wiki/images/3/3a/Gel_pic_pdawn.png)
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!
![](https://static.igem.org/mediawiki/2012/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg)
Figure 8