Difference between revisions of "Part:BBa K407008"
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<h2>Objective of part assembly</h2>
 
<h2>Objective of part assembly</h2>
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<p>In order to determine the kinetics and toxicity of the reporter proteins, ecfp and eyfp, constructs constitutively expressing both of them were assembled. Secondly, these fluorescent reporters are important tools to further improve and investigate our method of normalization like performed with RFP. Depending on the desired application and output signal, ecfp as well as eyfp can be coupled to our reporter systems described in the first characterized part.</p>
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<p>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.</p>  
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This part is meant to serve as a device that constitutively expresses the cyan fluorescent protein (CFP) by means of the promoter ptetR while repressing the same in the presence of tetracycline.  Hence, tetracycline acts as a negative regulator here.
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<p>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.</p>
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<p>Furthermore, this reporter system can additionally be used in any other system which is based on the lux operon.
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</p>
 
<h2>Materials and methods</h2>
 
<h2>Materials and methods</h2>
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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_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>
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<a href="https://static.igem.org/mediawiki/2010/8/81/PTetCFP_fluo3.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/8/81/PTetCFP_fluo3.jpg" class="border"></a>
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<div class="caption"><p>Figure 1:Light microscopy image of CFP fluroscence</p><p>of the assembled part</p>
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<p>The characterization was performed using overnight cultures of the corresponding part. The optical density of the cell cultures was adjusted to 0.4 and 50 µl of the culture sample was pipetted onto a microscope slide and a coverslip was placed over it. A 100X/1.3 Oil immersion objective was used for the light microscopy imaging and filters necessary for CFP fluorescence detection was set. An initial live imaging was done in transmitted light mode to find a field of view with maximum number of cells. The same was then used for capturing the fluorescence signal of the CFP. The same procedure was repeated several times in order to quantify the data. If we need to observe more number of cells under a given field of view, it is recommended to spin down the cell culture and use the resuspended pellet for increased cell density.</p>
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<h2>Results</h2>
 
<h2>Results</h2>
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<a href="https://static.igem.org/mediawiki/2010/d/dc/PTetCFP_fluo3_histogram.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/d/dc/PTetCFP_fluo3_histogram.jpg" class="border"></a>
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<a href="https://static.igem.org/mediawiki/2010/c/ce/Ptet_yfp.png"><img src="https://static.igem.org/mediawiki/2010/c/ce/Ptet_yfp.png" class="border right thumbs"></a>
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<div class="caption"><p>Figure 2:Histogram representing the fluorescence range of</p><p> CFP over an exposure time of 500ms</p>
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<div class="caption"><p>Figure 1: 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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<p>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.</p>
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<p>Figure 1 shows the fluorescence expression of the reporter protein CFP as it is expressed by itself by means of the repressible pTetR promoter.</p>
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Figure 2 represents the histogram of the image shown above. The fluorescence range of the CFP reporter can be seen in addition to the distribution of the experimental data over an exposure time of 500 ms.
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<h2>Discussion</h2>
 
<h2>Discussion</h2>
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<p>Figure 1 depicts the fluorescence observed by means of the light microscope using filters suitable for CFP excitation and emission spectra. E.coli cells that did not express any fluorescence were used as a negative control in order to detect any kind of background and distinguish them from the actual fluorescence.</p>
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<p>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.</p>
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<p>The fluorescence level of the CFP was clearly discernible and also reached saturation level when the cells were exposed for a longer period of time, say 1000 ms.</p>
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<p>From Figure 2, we can observe clearly the range of fluorescence and that the exposure time was optimal to avoid excessive saturation of the fluorescence signal. The mean value of fluorescence units observed.</p>  
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<h2>Additional information</h2>
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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 17:15, 7 November 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.