Team:METU Turkey/Characterization of an existing part

(Difference between revisions)

 

For parts BBa_I13521 and BBa_I13522, their excitation and emission maxima were determined. BBa_B0034 was used as a negative control. These data were used later in following characterization experiments as set parameters. The host was E. coli Top Ten in all experiments.


The graph above shows the emission scan for RFP in part BBa_I13521. Since it was the best option in terms of fluorescence yield, 570 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 605 nm.

Meanwhile, the graph below shows the emission scan for GFP in part BBa_I13522. Since it was the best option in terms of fluorescence yield, 395 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 515 nm.



The same procedure was also used for CFP in part BBa_I13602. However, the part did not work properly and gave a close fluorescence to our negative control.



For the parts BBa_I13521 and BBa_I13522 which are mentioned above, confocal laser scanning microscope images were taken in the host Top Ten.

While checking excitation and emission spectra, we also prepared a growth curve of Top Ten strain with BBa_I13522:



Our aim was to evaluate RFP expression quantity of E. coli. In order to obtain this information, we agreed that the measurement of fluorescence intensity in certain OD values is the best approach. However, fluorescence quenching was a setback which we must overcome for accurate and reliable results. Because, as a result of quenching, fluorescence intensity counts do not give the actual amount of fluorescence expected from a certain OD value. Therefore we employed a curve fitting method suggested by Zhang et. al (2010) which is claimed to take the quenching problem into consideration


Where If is the fluorescence intensity; OD is the cell density; y0 is the offset; A1 is the amplitude; and t1 is the decay constant.
We chose BBa_J04450 for this experiment. And our negative control was again BBa_B0034.
There were nine bacterial concentrations prepared for one to five hours of IPTG induction. (All concentrations were prepared as triplicates.) Then a complete graph of all the induction durations was plotted with the help of the function shown above:



As seen in the graph, fluorescence behaves exactly as expected and does not increase linearly with the increased bacterial concentration. However, IPTG induction effect is clear on fluorescence intensity. (Error bars were too small to be visible.)


Finally, the variables A1 and t1 of the fittings of each induction duration were used to obtain a mean fluorescence intensity (MFI) value:



By conducting this experiment, we were able to characterize RFP expression pattern of the part BBa_J04450 in the host Top Ten. Theoretical mean fluorescence intensity reflects the fluorescence quantum efficiency of the fluorophore RFP, and was found to be unrelated to the cell concentration. Since it is obvious from the results we have obtained that change in the OD does not correlate linearly with the change in the fluorescence intensity. Therefore this model proposes a solution to the unpredictability of the fluorescence amount coming from a certain bacterial population.