Team:Penn/BLSensor

From 2012.igem.org

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We then characterized the induction kinetics of the pDawn system through an mCherry expression time course.  We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 22 hours in a 37C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period.  After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD.  The cultures were then spun down in a centrifuge.  These results can be seen in Figure 3. </p>
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We then characterized the induction kinetics of the pDawn system through an mCherry expression time course.  We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 22 hours in a 37C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period.  After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD.  The cultures were then spun down in a centrifuge.  These results can be seen in Figure 2. </p>
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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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Revision as of 00:43, 27 October 2012

Penn 2012 iGEM Wiki

Image Map

YF1/FixJ (pDawn) Objectives

To characterize our pDawn gene expression system, we showed the following:
  1. pDawn allows for light-dependent gene expression in bacteria
  2. pDawn allows for light-dependent lysis of mammalian cells by bacteria
Light Dependent Gene Expression in Bacteria

We tested for light dependent gene expression by cloning in an mCherry reporter protein into the multiple cloning site of the pDawn system. First, we tested for the on-off ratio by growing cultures of BL21-pDawn-mCherry in both inducing and non-inducing conditions for 22 hours. After spinning down the cultures in a centrifuge, we were able to visually confirm the expression of mCherry due to the bacterial pellet grown in inducing conditions to be colored red, while the other pellet had no color (Figure 2)


Figure 1
Characterizing Time-Dependent Gene Expression

We then characterized the induction kinetics of the pDawn system through an mCherry expression time course. We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 22 hours in a 37C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period. After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD. The cultures were then spun down in a centrifuge. These results can be seen in Figure 2.



Figure 2
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 3