Team:BIOTEC Dresden/Characterized Parts/BBa K407008
Objective of part assembly
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.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.
Materials and methods
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.
Figure 1 shows the fluorescence expression of the reporter protein CFP as it is expressed by itself by means of the repressible pTetR promoter.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.
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.
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.
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.