Difference between revisions of "Part:BBa I13272"

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<h2>Part BBa_I13272</h2>  
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<a href="https://static.igem.org/mediawiki/2010/f/f5/I13272_%28Custom%29.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/f/f5/I13272_%28Custom%29.png" class="border left thumbs"></a>  
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<h2>BBa_I13263</h2>
<p>This part is supposed to serve as a potential detection and quantification system for the concentration of the signal molecule N-Acyl homoserine lactone (AHL) in the growth medium.  As shown in the parts overview LuxR is constitutively expressed. In the presence of AHL a LuxR-AHL complex is formed activating lux pR and thereby the expression of EYFP. Technically, it is the same part as BBa_K407014 but it has a different YFP-molecule as a reporter. In characterization experiments, the amount of AHL required for induction was analyzed. .</p>
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<a href="https://static.igem.org/mediawiki/2010/6/68/PartI13263_%28Custom%29.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/6/68/PartI13263_%28Custom%29.png" class="border thumbs left"></a>
<div class="visualClear"></div>
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<p>This part is supposed to serve as a potential detection and quantification system for the concentration of the signal molecule N-Acyl homoserine lactone (AHL) in the growth medium.  As shown in the parts overview LuxR is constitutively expressed. In the presence of AHL a LuxR-AHL complex is formed activating luxpR and thereby the expression of eyfp. In characterization experiments the amount of AHL required for induction was analyzed. This part is characterized best and was consequently always used for later experiments.</p>
<h3>Results</h3>
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<h3>Results</h3>
<a href="https://static.igem.org/mediawiki/2010/c/c3/Part18overt_%28Custom%29.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/c/c3/Part18overt_%28Custom%29.png" class="border thumbs"></a>
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  <div class="caption"><p>Figure 1: The fluorescence of YFP is shown over time</p>
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<a href="https://static.igem.org/mediawiki/parts/3/3a/17overt.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/parts/3/3a/17overt.png" class="border thumbs left"></a>
<p>for different AHL concentrations from 0 to 2000nM and for pure LB medium</p></div>
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<div class="caption"><p>Figure 1: The fluorescence of YFP is shown over time</p>
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<p>for different AHL concentrations from 0.1 to 2000nM and for pure LB medium</p></div>
<p>Figure 1 shows the development of the YFP signal against time for different AHL concentrations. Unexpectedly, the results of the measurements vary greatly from the data we achieved with the part BBa_I13272 that is identical to this part except that it contains a different YFP. Nevertheless, there is an increase in fluorescence signal with increasing AHL concentration. Starting from an AHL concentration of 500nM. The LB medium has a fluorescent signal, which remains almost constant during the measurement.</p>
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<p>The same trend can be seen in Figure 2, which displays the fluorescence over increasing AHL concentrations after a two hours incubation time. Here again, the curve looks similar to the one of part BBa_I13272 but the plateau is not as nicely visible.</p>
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<p>The Figure 1 shows the YFP development over time of different concentrations of AHL, ranging from 10pM to 2µM.  It can be seen that the fluorescence signal does not significantly change over time for AHL concentrations below 100nM and that the amount of YFP produced is the same for all concentrations above 300nM.</p>
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<p>In Figure 2 the fluorescence as a function of the AHL concentration after two hours of incubation is visualized. It can be seen that the YFP signal after 2 hours of incubation increases with increasing AHL concentrations but reaches a plateau at about 700nM. </p>
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<h3>Discussion:</h3>
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  <a href="https://static.igem.org/mediawiki/2010/9/92/Part17overC_%28Custom%29.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/92/Part17overC_%28Custom%29.png" class="border thumbs left"></a>
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<div class="caption"><p>Figure 2: The fluorescence of YFP is shown</p>
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<p>over increasing AHL concentrations after an icubation time of 2h</p></div>
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<p>Figure 1 shows nicely that the LB medium (dark blue) has a fluorescence signal of about 2800 raw fluorescence units (RFU). But since the fluorescence signal stayed constant over the time measured, this fluorescence does not qualitatively influence the data and can thus be ignored. Additionally it can be seen that the concentrations below 100nM do not lead to an increase in fluorescence, leading to the conclusion that a minimum concentration of 100nM outside of the bacteria is required for the induction of the LuxpR and in turn for the detectable expression of YFP. Furthermore, the graph shows that after 2h of incubation with AHL the amount of YFP detected can directly by related to the initial concentration of AHL. </p>
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<p>Figure 2 on the other hand shows a logarithmic behavior of the transcriptional induction of the LuxpR promoter by AHL, which reaches its maximum induction at an AHL concentration of about 750nM. </p>
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<div class="visualClear"></div>
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<h3>Materials and methods:</h3>
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<p>The characterization was performed using a 96-well plate and a fluorescence plate reader. Bacteria supplied with the part BBa_I13263 were suspended in medium of a certain concentration of AHL ranging from 0.01 to 2000nM. The fluorescence was measured every 5 minutes using an excitation wavelength of 485nm and an emission wavelength of 535nm. For every fluorescence value, also the optical density at 612nm was measured. As a negative control the same measurements were done on uninduced bacteria and LB-medium without cells.</p>
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<h3>Additional information</h3>
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<p>For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki <a href="https://static.igem.org/mediawiki/igem.org/7/75/Protocol_AHLassay.pdf"> here.</a In case you want to know more about the normalization and negative controls, please have a look at this page: (Link)</p>
  
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<a href="https://static.igem.org/mediawiki/2010/f/f8/Part18overC_%28Custom%29.png" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/f/f8/Part18overC_%28Custom%29.png" class="border left thumbs"></a>
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  <div class="caption"><p>Figure 2: The fluorescence of YFP is shown over increasing</p>
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<p> concentrations of AHL after 2 hours of incubation</p></div>
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<h3>Discussion:</h3>
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<p>The outcome of this experiment was unexpected regarding the same measurement of the part BBa_I13272. This part was expected to show the exact same behavior. It is difficult to explain the discrepancies. Since the only differences between these two parts is the yellow fluorescent protein encoded, it must be concluded that the EYFP (E0034) is either not as well transcribed or translated as the eyfp (E0030) or it is not as functional. Mutations in the coding sequence can lead to an decreased expression correlating to decreased fluorescence signal. </p>
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<div class="visualClear"></div>
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<h3>Materials and methods:</h3>
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<p>The characterization was performed using a 96-well plate and a fluorescence plate reader, which was kept at 37°C during the whole measurement.. Bacteria supplied with the part BBa_I13272 were suspended in medium of a certain concentration of AHL ranging from 0.01 to 2000nM. The fluorescence was measured every 5 minutes using an excitation wavelength of 485nm and an emission wavelength of 535nm. For every fluorescence value, also the optical density at 612nm was measured. As a negative control the same measurements were done on uninduced bacteria and LB-medium without cells.</p>
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<h3>Additional information</h3>
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<p>For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki <a href="https://static.igem.org/mediawiki/igem.org/7/75/Protocol_AHLassay.pdf"> here.</a></p>
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Revision as of 16:42, 7 November 2010

BBa_I13263

This part is supposed to serve as a potential detection and quantification system for the concentration of the signal molecule N-Acyl homoserine lactone (AHL) in the growth medium. As shown in the parts overview LuxR is constitutively expressed. In the presence of AHL a LuxR-AHL complex is formed activating luxpR and thereby the expression of eyfp. In characterization experiments the amount of AHL required for induction was analyzed. This part is characterized best and was consequently always used for later experiments.

Results

Figure 1: The fluorescence of YFP is shown over time

for different AHL concentrations from 0.1 to 2000nM and for pure LB medium

The Figure 1 shows the YFP development over time of different concentrations of AHL, ranging from 10pM to 2µM. It can be seen that the fluorescence signal does not significantly change over time for AHL concentrations below 100nM and that the amount of YFP produced is the same for all concentrations above 300nM.

In Figure 2 the fluorescence as a function of the AHL concentration after two hours of incubation is visualized. It can be seen that the YFP signal after 2 hours of incubation increases with increasing AHL concentrations but reaches a plateau at about 700nM.

Discussion:

Figure 2: The fluorescence of YFP is shown

over increasing AHL concentrations after an icubation time of 2h

Figure 1 shows nicely that the LB medium (dark blue) has a fluorescence signal of about 2800 raw fluorescence units (RFU). But since the fluorescence signal stayed constant over the time measured, this fluorescence does not qualitatively influence the data and can thus be ignored. Additionally it can be seen that the concentrations below 100nM do not lead to an increase in fluorescence, leading to the conclusion that a minimum concentration of 100nM outside of the bacteria is required for the induction of the LuxpR and in turn for the detectable expression of YFP. Furthermore, the graph shows that after 2h of incubation with AHL the amount of YFP detected can directly by related to the initial concentration of AHL.

Figure 2 on the other hand shows a logarithmic behavior of the transcriptional induction of the LuxpR promoter by AHL, which reaches its maximum induction at an AHL concentration of about 750nM.

Materials and methods:

The characterization was performed using a 96-well plate and a fluorescence plate reader. Bacteria supplied with the part BBa_I13263 were suspended in medium of a certain concentration of AHL ranging from 0.01 to 2000nM. The fluorescence was measured every 5 minutes using an excitation wavelength of 485nm and an emission wavelength of 535nm. For every fluorescence value, also the optical density at 612nm was measured. As a negative control the same measurements were done on uninduced bacteria and LB-medium without cells.

Additional information

For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki here.