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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:
The different constructs used are:
- p[F]::mKATE, also referred here as LD2
- p[J]::mKATE, also referred here as LD3
- p[F] incl. N-enzymes, also referred here as LD4
- p[J] incl. N-enzymes, also referred here as LD5
- p[F]::mKATE p[J]::mKATE, also referred here as LD6
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 LD2 (p[F]::mKATE) and LD3 (P[J]::MKATE), different concentrations of 2,4-DNT were tested:
The BioBricks showed an increasing fluorescence signal over time when they were induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescence signal. The construct LD2 (p[F]::mKATE) (sample B on the Figures) showed a much higher response than LD3 (P[J]::MKATE) (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature [1]. The induced negative control (sample D on the Figures) shows no fluorescence, which indicates that the yqjF promoter is positively induced by 2,4-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 attained 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 LD2 (p[F]::mKATE) after several hours of their induction with DNT (figure 2 bottom) compared to the early stages of induction (figure 2 top). Figure 3 clearly shows the increase in fluorescence signal of the two cultures carrying the LD2 (p[F]::mKATE) 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.
