Team:BIOTEC Dresden/Characterized Parts/BBa I13272

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<h3>Additional information</h3>
<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="http://igem.org/wiki/images/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> 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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<p>For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki <a href="http://igem.org/wiki/images/7/75/Protocol_AHLassay.pdf"> here.</a></p>
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[[Category:BIOTEC_Dresden/Characterized_Parts|I13272]]
[[Category:BIOTEC_Dresden/Characterized_Parts|I13272]]
{{Biotec_Dresden/Bottom}}
{{Biotec_Dresden/Bottom}}

Latest revision as of 02:37, 28 October 2010

Part BBa_I13272

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 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.

Figure 1: The fluorescence of YFP is shown over time

for different AHL concentrations from 0 to 2000nM and for pure LB medium

Figure 2: The fluorescence of YFP is shown over increasing

concentrations of AHL after 2 hours of incubation

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.


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