Team:TU Delft-Leiden/WetLab/landmine/characterisation

Landmine Module - Characterisation

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 at

LINK TO PROTOCOLS

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

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.