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Team:TU Delft-Leiden/WetLab/landmine/characterisation
From 2014.igem.org
Module Landmine Detection – Characterization
In the wet lab, we made constructs containing the fluorescent protein mKate2 regulated by the ybiJ and yqjF promoters, which are thought to be activated with some chemicals such as 2,4-DNT or 1,3-DNB. Here you can find information with respect to the characterization of the BioBricks for the Landmine Detection module.
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Module Landmine Detection
- Context
- Integration of Departments
- Cloning
- Characterization
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, which corresponds to BioBrick BBa_K1316003
- p[J]::mKATE, also referred here as LD3, which corresponds to BioBrick BBa_K1316005
- p[F] incl. N-enzymes, also referred here as LD4, which corresponds to BioBrick BBa_K1316007
- p[J] incl. N-enzymes, also referred here as LD5, which corresponds to BioBrick BBa_K1316008
- p[F]::mKATE p[J]::mKATE, also referred here as LD6, which corresponds to BioBrick BBa_K1316009
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). 2,4-DNT, due to safety reasons, was bought in liquid solution dissolved in acetonitrile. To test that the compound triggering the response of the promoters is 2,4-DNT and not acetonitrile, the 0 mg/L 2,4-DNT solution was prepared with the same concentration of acetonitrile contained in the most concentrated 2,4-DNT solution.
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 2,4-DNT (same acetonitrile concentration as the 100 mg/L 2,4-DNT sample) did not kill the cells. 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 are induced with L-Rhamnose, the 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).
It could still be that at 50 mg/L 2,4-DNT, what triggers the response of the promoters is actually the acetonitrile and not the 2,4-DNT, but that would be strange because at 0 mg/L 2,4-DNT (acetonitrile is 2 times more concentrated than at 50 mg/L 2,4-DNT) the cells survive and there is no fluorescence response.
This data suggests that both promoters, ybiJ and yqjF, respond to 2,4-DNT; and that the presence of the N-genes is of high importance for this response. Hence, we conclude 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 in order to obtain a good result the N-genes must be expressed.
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 was used to test the LD2 (p[F]::mKATE) construct. Cultures of this construct were induced with 50 mg/L 2,4-DNT. Aliquots of the culture were taken after 2 and 6 hours; centrifuged and resuspended in PBS solution to eliminate the 2,4-DNT and the acetonitrile; and were then analysed at the FACS machine. At the same time, a strain constitutively expressing mKate2 and an empty strain were used as positive and negative controls respectively. Two cultures of LD2 were used (samples 1 and 2 in figure 2) in order to get the LD2 data in duplo.
The results of the FACS experiments show that the fluorescent response increases with time after induction and, therefore, it means that the yqjF promoter was activated. Figure 2 contains all the LD2 culture (2 and 6 hours after induction), as well as the positive and negative controls. Figure 3 only contains the LD2 culture (2 and 6 hours after induction) and, hence, it provides a more clear picture of the promoter induction. Unfortunately, no solution was prepared containing acetonitrile and water alone and, therefore, we cannot conclude that the induction was caused by 2,4-DNT as it could have been caused by acetonitrile. Nevertheless, as aforementioned, the plate reader results suggests that 2,4-DNT is more likely to be the inducer of the promoters rather than acetonitrile.
Results - FACS
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.
