Team:Penn/BLSensor

From 2012.igem.org

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<b><div class="name" align="center">YF1/FixJ Characterization</div></b>
<b><div class="name" align="center">YF1/FixJ Characterization</div></b>
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<p style="color:black;text-indent:30px;">After reading a lot of papers to select an appropriate light sensing system to use, we selected the YF1/FixJ blue light system. We were also considering the red light sensor Cph8 but ultimately decided on YF1/FixJ because of its high on/off gene expression and also because of its availability to us (we were fortunate enough to come across the YF1/FixJ system in the form of the pDawn plasmid from the Moglich lab in Germany.)</p>
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<p style="color:black;text-indent:30px;">After reading many papers to select an appropriate light-sensing system to use, we selected the YF1/FixJ blue light system. We had also considered the red light sensor Cph8 but ultimately decided on YF1/FixJ because of its high on/off ratio of gene expression and also because of its availability to us (we were fortunate enough to come across the YF1/FixJ system in the form of the pDawn plasmid from the Moglich lab in Germany).</p>
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<b><div class="name" align="center">YF1/FixJ System (pDawn)</div></b>
<b><div class="name" align="center">YF1/FixJ System (pDawn)</div></b>
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<p style="color:black;text-indent:30px;">Shown below in Figure 1, the YF1/FixJ system works through a "repress the repressor" concept. Upon 480 nm blue light illumination, YF1 (a fusion of a LOV protein domain and a histidine kinase) phosphorylates a FixJ response regulator that activates the pFixK2 promoter. The activation of pFixK2, promotes expression of the cI repressor that in turn represses the lambda promoter pR. The net result is activation of the gene in the downstream MCS. </p>
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<p style="color:black;text-indent:30px;"> As shown below in Figure 1, the YF1/FixJ system works through a "repress the repressor" concept. Upon 480 nm blue light illumination, YF1 (a fusion of a LOV protein domain and a histidine kinase) phosphorylates a FixJ response regulator that activates the pFixK2 promoter. The activation of pFixK2, promotes expression of the cI repressor that, in turn, represses the lambda promoter pR. The net result is activation of the gene in the downstream MCS. </p>
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<div align="center"><img src="http://2012.igem.org/wiki/images/7/74/PDAWN.gif" width="500" height="300" />
<div align="center"><img src="http://2012.igem.org/wiki/images/7/74/PDAWN.gif" width="500" height="300" />
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To characterize our pDawn gene expression system, we showed the following:
To characterize our pDawn gene expression system, we showed the following:
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<li> pDawn allows for light-dependent gene expression in bacteria
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<li> pDawn allows for light-dependent gene expression in bacteria.
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<li> pDawn allows for light-dependent lysis of mammalian cells by bacteria
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<li> pDawn allows for light-dependent lysis of mammalian cells by bacteria.
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<b><div class="name" align="center">Light Dependent Gene Expression in Bacteria</div></b>
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<b><div class="name" align="center">Light-Dependent Gene Expression in Bacteria</div></b>
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<br>
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<p style="color:black;text-indent:30px;">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)</p>
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<p style="color:black;text-indent:30px;">We tested for light-dependent gene expression by cloning 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).</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 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, and 22 hours in a 37°C 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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<div align="center">
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Revision as of 20:58, 26 October 2012

Penn 2012 iGEM Wiki

Image Map

YF1/FixJ Characterization

After reading many papers to select an appropriate light-sensing system to use, we selected the YF1/FixJ blue light system. We had also considered the red light sensor Cph8 but ultimately decided on YF1/FixJ because of its high on/off ratio of gene expression and also because of its availability to us (we were fortunate enough to come across the YF1/FixJ system in the form of the pDawn plasmid from the Moglich lab in Germany).

YF1/FixJ System (pDawn)

As shown below in Figure 1, the YF1/FixJ system works through a "repress the repressor" concept. Upon 480 nm blue light illumination, YF1 (a fusion of a LOV protein domain and a histidine kinase) phosphorylates a FixJ response regulator that activates the pFixK2 promoter. The activation of pFixK2, promotes expression of the cI repressor that, in turn, represses the lambda promoter pR. The net result is activation of the gene in the downstream MCS.



Figure 1
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 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 2
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, and 22 hours in a 37°C 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.



Figure 3