Difference between revisions of "Part:BBa I13263"
| Line 18: | Line 18: | ||
</script> | </script> | ||
</head> | </head> | ||
| − | + | <div id="content_prim"> | |
| − | <h2> | + | <h2>Part BBa_I13272</h2> |
| − | <a href="https://static.igem.org/mediawiki/2010/ | + | <a href="https://static.igem.org/mediawiki/2010/f/f5/I13272_%28Custom%29.png" rel="lightbox" class="border left thumbs"><img src="https://static.igem.org/mediawiki/2010/f/f5/I13272_%28Custom%29.png" class="border left"></a> |
| − | <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 | + | |
| + | <div class="border right thumbs"> | ||
| + | <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 left"></a> | ||
| + | <div class="caption"><p>Figure 1: The fluorescence of YFP is shown over time</p> | ||
| + | <p>for different AHL concentrations from 0 to 2000nM and for pure LB medium</p></div> | ||
| + | </div> | ||
| + | <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> | ||
<div class="visualClear"></div> | <div class="visualClear"></div> | ||
<h3>Results</h3> | <h3>Results</h3> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | <p>The Figure 1 shows the | + | <div class="border left thumbs"> |
| − | <p> | + | <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"></a> |
| + | <div class="caption"><p>Figure 2: The fluorescence of YFP is shown over increasing</p> | ||
| + | <p> concentrations of AHL after 2 hours of incubation</p></div> | ||
| + | </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> | ||
| + | <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> | ||
<div class="visualClear"></div> | <div class="visualClear"></div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <h3>Discussion:</h3> | ||
| + | |||
| + | <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> | ||
| + | |||
| + | <h3>Materials and methods:</h3> | ||
| + | <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> | ||
| + | |||
| + | <h3>Additional information</h3> | ||
| + | <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> | ||
<hr> | <hr> | ||
</div></body> </html> | </div></body> </html> | ||
Revision as of 17:02, 7 November 2010
Part BBa_I13272
Figure 1: The fluorescence of YFP is shown over time
for different AHL concentrations from 0 to 2000nM and for pure LB medium
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. .
Results
Figure 2: The fluorescence of YFP is shown over increasing
concentrations of AHL after 2 hours of incubation
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.
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.
Discussion:
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.
Materials and methods:
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.
Additional information
For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki here.