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Team:BIOTEC Dresden/Characterized Parts/BBa K407013
From 2010.igem.org
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<h2>Materials and methods</h2> | <h2>Materials and methods</h2> | ||
| - | <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 | + | <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_407013 were suspended in medium of a certain concentration of AHL of 500 nM and 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> |
<h2>Results</h2> | <h2>Results</h2> | ||
<div class="border left"> | <div class="border left"> | ||
Revision as of 03:09, 28 October 2010
Objective of part assembly
To detect the amount of AHL present in the extracellular environment, different detection systems were built which assure a high sensitivity. For this purpose existing parts from the registry were assembled.
Two constructs were assembled that constitutively express LuxR in the detector cell. Downstream of the construct, a LuxR sensitive promoter is situated which is activated when AHL is present in the detector cell. An AHL-LuxR complex forms and binds to the luxR promoter thereby activating the expression of the reporter protein. As reporter protein; eyfp (yellow fluorescent protein) was used. Therefore, the assembled parts respond to AHL input. In SensorBricks, the LuxI enzyme is present outside the cell transforming SAM into AHL. Fluorescent measurements thereby correlate to the quantity of detected antigen in the sample.
Furthermore, this reporter system can additionally be used in any other system which is based on the lux operon.
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_407013 were suspended in medium of a certain concentration of AHL of 500 nM and 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.
Results
Figure 1: The fluorescence of YFP is shown over increasing
concentrations of AHL after 2 hours of incubation
The above figure shows the plot between the Raw Fluorescence Units and Time in minutes for the Lux receiver which has a YFP output when induced by different AHL concentrations such as 500 nM and 2000 nM. From the graph, it is evident that there is an increasing fluorescence signal with increase in time of measurement. In case of higher levels of AHL concentration, the signal output is also seen on the higher scale. The LB medium has a fluorescent signal, which remains almost constant during the measurement.
Discussion
Only two concentrations, viz. 500 Nm and 2000 nM of AHL were tested for induction of the cells which does not totally quantify the data which we have. Hence, it would be useful to test the same part at really low concentrations of AHL and determine the level of fluorescence output received. At zero concentration of AHL, a slight amount of fluorescence was detected which might be due to one of the following reasons: (i) The part was tested in a plasmid backbone containing ampicillin wherein there were no stop codons at the end of the construct. (ii) The promoter might have been leaky. This holds further scope as it would make sense to test the part in chloramphenicol backbone so as to strongly repress the constitutive expression of the reporter protein.
Additional information
For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki here.
