Team:Penn/LightActivatedLysis

From 2012.igem.org

(Difference between revisions)
Line 50: Line 50:
<div class="bigbox">
<div class="bigbox">
-
<b><div class="name" align="center">Verification of expression of Cytolysin A (ClyA)</div></b><br />
+
<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>
<br>
<br>

Revision as of 13:14, 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 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 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
pDawn and Nissle 1917

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