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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 different final Landmine detection constructs LD2-6, different concentrations of 2,4-DNT were tested (figure 1). The fact that the positive control (constitutively expressed mKate2) presents a clear fluorescence signal at 0 and 50 mg/L, but not at 100 mg/L indicates that high concentrations of 2,4-DNT are toxic for cells after several hours. The toxic compound seems to be 2,4-DNT and not acetonitrile because the sample at 0 mg/L DNT has the same acetonitrile concentration as the 100 mg/L DNT sample. Constructs LD2, LD3 and LD6 as well as LD4 and LD5 with no induction of the N-genes show no clear mKate2 induction over time. Not many conclusions can be drawn form LD2 due to the big standard deviation. However, when the N-genes, constructs LD4 and LD5 showed a clear increase in fluorescence over time for a concentration of 50 mg/L 2,4-DNT. In this situation, besides, LD5 seems to be more sensitive, as there is less leakage (there is less fluorescence signal at 0 mg/L DNT).From this data we concluded that the two best BioBricks for Landmine detection are (in this order) LD5 (p[J] incl. N-enzymes) and LD4 (p[F] incl. N-enzymes), and to obtain a good result the N-genes must be expressed.
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.
