Team:PennState/diauxie/progress

From 2008.igem.org

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<p class="start">We have completed our first set of cloning phases and are conducting our first round of testing and modification. Each test construct (promoter + GFP) has been cloned into pSB1A2 and transformed into DH5α, W3110 ∆xylB-G, and W3110 ∆xylB-R. Preliminary induction studies were run to find the optimal induction time and to see in what range OD and fluorescence were linearly related.</p>
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<p class="start">Each test construct (promoter + GFP) was cloned into the pSB1A2 plasmid and transformed into several <em>E. coli</em> strains: DH5α, W3110 ∆xylB-G, and W3110 ∆xylB-R. Preliminary induction studies were run to find the optimal induction time and to analyze the linear range for OD versus fluorescence.</p>
<h6>Test Construct</h6>
<h6>Test Construct</h6>
<img src="https://static.igem.org/mediawiki/2008/1/1b/Test_construct.JPG" alt="[Test Consturct]" title="" style="width: 100%; border: solid 1px #000" />
<img src="https://static.igem.org/mediawiki/2008/1/1b/Test_construct.JPG" alt="[Test Consturct]" title="" style="width: 100%; border: solid 1px #000" />
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<table style="text-align: center; width: 100%;" class="progresschecker">
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<p>Tests were then run to compare the levels of induction with various mixes of xylose, glucose and xylose+glucose. The goal was to obtain a noticeably higher level of induction with the xylose/glucose mixture when compared to the wild-type construct.</p>
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  <td width="20%">Promoter</td>
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  <td width="20%">pSB1A2</td>
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  <td width="20%">DH5&alpha;</td>
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  <td width="20%">W3110 &Delta;xylB-G</td>
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  <td width="20%">W3110 &Delta;xylB-R</td>
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  <td style="font-weight: bold">PN</td>
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  <td style="font-weight: bold">P1</td>
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  <td style="font-weight: bold">P3</td>
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<p>Test were then run to compare the level of induction between mixes of xylose, glucose and xylose, and glucose. The goal of this project is to obtain a noticeably higher level of induction when induced with xylose and glucose compared to the wildtype construct.</p>
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   <div class="gallerytext"><p>DH5a fluoresence</p></div>
   <div class="gallerytext"><p>DH5a fluoresence</p></div>
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   <div class="gallerytext"><p>W3110 fluoresence</p></div>
   <div class="gallerytext"><p>W3110 fluoresence</p></div>
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<p>These graphs show the normalized fluoresence strength for PN, P1, and P3 xylose promoters induced with xylose, glucose, and a mixture.  The W3110 cells have <em>xylE</em> and <em>xylG</em> knocked out while DH5α still contain the natural xylose transport and metabolim.  This data shows that there is little effect on the fluorescence intensity using strains with <em>xylE</em> and <em>xylG</em> sequences deleted.  Our next step is to transform these promoters into <em>E. coli</em> cells with deleted xylose metabolism and transporters. </p>
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   <div class="gallerytext"><p>P3 induction time</p></div>
   <div class="gallerytext"><p>P3 induction time</p></div>
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<p>The cells were induced iduced with sugars and then allowed to grow with sample being removed every half hour.  The goal was to find the induction time where fluorescence starts to level off.  The intensity has leveled off and begins to drop after 7.5 hours so this was the growth time for all future tests.
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   <div class="gallerytext"><p>P1 linear range</p></div>
   <div class="gallerytext"><p>P1 linear range</p></div>
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   <div class="thumb" style="padding: 18px 0pt; width: 150px;"><div style="margin: auto; width: 120px;"><a href="/Image:P3_linear_penn_state08.jpg" class="image" title="P3_linear_penn_state08.jpg"><img alt="" src="/wiki/images/f/f5/P3_linear_penn_state08.jpg" border="0" height="220" width="240"></a></div></div>
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   <div class="thumb" style="padding: 18px 0pt; width: 150px;"><div style="margin: auto; width: 120px;"><a href="/Image:P3_linear_penn_state08.jpg" class="image" title="P3_linear_penn_state08.jpg"><img alt="" src="/wiki/images/f/f5/P3_linear_penn_state08.jpg" border="0" height="110" width="120"></a></div></div>
   <div class="gallerytext"><p>P3 linear range</p></div>
   <div class="gallerytext"><p>P3 linear range</p></div>
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<p>The three promoters analyzed show linear behavior in the optical density range.  The strength of fluorescence did change depending on the promoter.  With this information we were able to accurately normalize fluorescence data.
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Latest revision as of 02:19, 30 October 2008

Home The Team The Project Parts Notebook

Diauxie Elimination

Introduction
The System
Strategies
Progress
Conclusions
Parts
References

NHR Biosensors

NHR Introduction
Phthalate Biosensor
BPA Biosensor
 Progress & Results

Each test construct (promoter + GFP) was cloned into the pSB1A2 plasmid and transformed into several E. coli strains: DH5α, W3110 ∆xylB-G, and W3110 ∆xylB-R. Preliminary induction studies were run to find the optimal induction time and to analyze the linear range for OD versus fluorescence.

Test Construct
[Test Consturct]

Tests were then run to compare the levels of induction with various mixes of xylose, glucose and xylose+glucose. The goal was to obtain a noticeably higher level of induction with the xylose/glucose mixture when compared to the wild-type construct.

DH5a fluoresence

W3110 fluoresence

These graphs show the normalized fluoresence strength for PN, P1, and P3 xylose promoters induced with xylose, glucose, and a mixture. The W3110 cells have xylE and xylG knocked out while DH5α still contain the natural xylose transport and metabolim. This data shows that there is little effect on the fluorescence intensity using strains with xylE and xylG sequences deleted. Our next step is to transform these promoters into E. coli cells with deleted xylose metabolism and transporters.

PN induction time

P1 induction time

P3 induction time

The cells were induced iduced with sugars and then allowed to grow with sample being removed every half hour. The goal was to find the induction time where fluorescence starts to level off. The intensity has leveled off and begins to drop after 7.5 hours so this was the growth time for all future tests.

PN linear range

P1 linear range

P3 linear range

The three promoters analyzed show linear behavior in the optical density range. The strength of fluorescence did change depending on the promoter. With this information we were able to accurately normalize fluorescence data.

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