Team:TU Delft-Leiden/WetLab/landmine

Module ‘Landmine Detection'

Described are the general why and how of the practices Modeling as well as Microfluidics with respect to experimental work performed in the module Landmine Detection.

Integration of Modeling, Experimental Work and Microfluidics

Escherichia coli is the model organism in which a system is implemented that is able to trigger a reaction in and of this organism to the presence of compounds leeching from landmines. Escherichia coli carry promoters of genes hypothesized to respond to compounds containing nitrogen. DNT, TNT and DBP contain this element and leech from landmines. By coupling of fluorescent molecule mKATE2 to (improved versions of) these promoters, qualification of the response and hence of the presence of the compound is possible. This module thus encompasses the development of a biosensor.

Modeled are the responses of Escherichia coli to compounds DNT, TNT and DNB in a straightforward fashion. Input are compounds, generated is the response of the promoter; datapoints resulting from imaging mKATE2 fluorescence and linking fluorescence to compounds added at defined points in time after induction with relevant compounds. Black box in this Module are the regulatory enzymes, possibly nitrogen-linked, and genes encoding these elements.

Experimental work centers on responsive promoters, compounds, and, in addition, response depending on several enzymes functional in nitrogen metabolism in order to shine light on the black box regarding the reaction and actual molecular basis of reaction to compounds leeching from landmines.

Implementation of a system of microfluidics within this Module is based on the aspiration to be able to perform in-field measurements of compounds leeching from landmines. The necessity of having a certain type of device points towards use microfluidics. Aspects central in development of this type of device are, amongst others, the speed of reactions, the possibility of in vivo measurements at all points in time and options for quantification.

Aim, construction and functionality of what is hereafter referred to as the mother machine are discussed under [.]. This type of microfluidic system is intended for single-cell experiments, utilizing nanoscale channels coupled to a central channel for flow of media. Team iGEM 2014 TU Delft – Leiden University has made use of the standard model developed at Massachusetts Institute of Technology.

Summarizing, the device is constructed via positive silicon waver coupled to a plastic mold. The team has constructed its negative with PDMS, generating channels. Size of channels and size of cells should be taken in consideration, the latter partly depending on growth conditions. Holes and tubes coupled to these holes, coupled to the central channel deal with flushing with medium, with respect to the Module Landmine Detection carrying compounds DNT, TNT or DNB. Before application, the PDMS sample is connected to a glass slide by oxygenplasma and prepared for action by consecutive flushings with, amongst others, BSA. [imagen] More information regarding the design and construction of the mother machine can be found in the section [.].

Background Information

On the literature [1] a very interesting type of biosensor was found: two natural promoters of Escherichia coli (ybiJ and yqjF) were found to be activated in the presence of some aromatic N-based compounds such as 2,4,6-Trinitrotoluene (2,4,6-TNT), 2,4-Dinitrotoluene (2,4-DNT) and 1,3-Dinitrobenzene (1,3-DNB). Land mines are mainly composed of 2,4,6-TNT, but many times impurities of 2,4-DNT and 1,3-DNB are also present. These last two compounds are more volatile than 2,4,6-TNT and, therefore, they can more easily leak out of the land mine. As a consequence, Belkin and co-workers envisioned in the two aforementioned promoters (ybiJ and yqjF) a high potential to develop a biosensor for land mine detection.

This was a very attractive case of study to implement the Plug-and-Play biosensor based on electrical current developed by our team.

Coupling the promoters to our system

As a final goal, it was desired to link the ybiJ and yqjF promoters to the generation of an electrical current. Therefore, it was our intention to express two of the three genes of the mtrCAB pathway independently (either constitutively or regulated via an inducer) and the last mtrCAB gene regulated by one of the two promoters induced by land mine compounds. Hence, the presence of land mines would trigger the expression of the lacking gene of the mtrCAB pathway, and therefore, it would start the secreation of electrons out of the cell. This way, it was intended to correlate the presence of the abovementioned chemicals present in land mines with a certain electrical current.

It was, nevertheless, more convenient to test the selected promoters with a more easily measurable reporting system

Choosing a reporter gene

To initially check the functionality of the ybiJ and yqjF promoters, a fluorescent protein mKate2 was chosen as a reporter. mKate2 was selected instead of the conventional Green Fluorescent Protein (GFP) because it has been observed to be more evenly expressed among the different cells of a cellular culture than GFP.

Cloning Strategy and Characterisation of this module

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.