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Team:TU Delft-Leiden/WetLab/landmine/characterisation
From 2014.igem.org
Module Landmine Detection - Characterization
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As already mentioned, the promoters found to be activated in presence of several chemical compounds that can leak from land mines (ybiJ and yqjF) were coupled to the expression of the fluorescent protein mKate2.
Assays
The different assays used to test our Land Mine BioBricks are:
Plate Reader
Microscopy
FACS
Plate Reader
A plate reader is a machine designed to handle samples on 6-1536 well format microtiter plates for the measuring of physical properties such as absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarisation. Concerning this module, the plate reader device was used for the measurement of fluorescence intensity generated by cells carrying the BioBricks designed to detect land mines. The final protocol developed for Plate reader analysis for this module can be found by clicking on this link
.Results - Plate Reader
Using the constructs BBa_K1316003 and BBa_K1316005, different concentrations of 2,4-DNT were tested:
The BioBricks showed an increasing fluorescent signal over time when they were induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescent signal. The BioBrick BBa_K1316003 (sample B on the Figures) showed a much higher response than BBa_K1316005 (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature [1]. The non-induction of the negative control (sample D on the Figures) indicates that it is the presence of the promoter that generates the signal in front of the presence of DNT.
Microscopy
FACS
Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry that allows the separation of individual cells based on the specific light scattering and fluorescent characteristics of each cell. Using FACS, information can be known of the size, shape and fluorescence of individual cells, therefore, it is a technique that can be used to observe the fluorescent response of our Landminde detection BioBricks in front of DNT.
The FACS technology allows us to see that, per cell, more fluorescence is produced by the construct BBa_K1316003 after several hours of their induction with DNT (figure 2 bottom) compared to the early states of induction (figure 2 top). Figure 3 clearly shows the increase in fluorescent signal of the two cultures carrying the BBa_K1316003 BioBrick.
Conclusions
From the assays performed it can be concluded that:References
[1] S. Yagur-Kroll, S. Belkin et al., “Escherichia Coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene”, Appl. Microbiol. Biotechnol. 98, 885-895, 2014.
