"
Team:Penn/LightActivatedLysis
From 2012.igem.org
| Line 9: | Line 9: | ||
.pic1{ float:left; margin:0 40px 0 0; width:150px;} | .pic1{ float:left; margin:0 40px 0 0; width:150px;} | ||
.name{ font-size:20px;} | .name{ font-size:20px;} | ||
| + | .figs2{width:916px; margin:0 auto; overflow:hidden;} | ||
| + | .fignew{font-size:13px; width:418px; margin:10px auto; float:left; padding:0 20px 0 20px;} | ||
.fig{font-size:13px; width:500px; margin:10px auto;} | .fig{font-size:13px; width:500px; margin:10px auto;} | ||
</style> | </style> | ||
| Line 18: | Line 20: | ||
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 2.</p> | 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 2.</p> | ||
| - | <div class=" | + | <div class="figs2"> |
| - | < | + | <div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/4/42/PDawn-mCherry-Dark-Grey-BG.jpg" height="250"><br> |
| + | </div>pDawn-mCherry Dark</div> | ||
| + | |||
| + | <div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/c/c0/PDawn-mCherry-Light-Grey-BG.jpg" height="250"><br> | ||
| + | </div>pDawn-mCherry Light</div></div> | ||
| + | <br> | ||
| + | <div class="figs2"> | ||
| + | <div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/d/db/PDawn-ClyA-His-Dark-Grey-BG.jpg" height="250"><br> | ||
| + | </div>pDawn-His-ClyA Dark</div> | ||
| + | |||
| + | <div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/3/35/PDawn-ClyA-His-Light-Grey-BG.jpg" height="250"><br> | ||
| + | </div>pDawn-His-ClyA Light</div></div> | ||
| + | |||
| + | |||
</div> | </div> | ||
<div class="bigbox"> | <div class="bigbox"> | ||
Revision as of 10:00, 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 2.
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 5
