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

Context

In 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 [1] 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, the red fluorescent protein, mKate2, was chosen as a reporter. mKate2 was selected instead of the conventional Green Fluorescent Protein (GFP) for two reasons. On one hand, illuminating cells with red light is less toxic than with blue/ green light. On the other hand, mKate2 is knwon to produce very intense fluorescence.

Possible improvement in the Landmine Detectionsystem

nfsA, nfsB and nemA are the genes for NfsA, NfsB and NEM reductases respectively. In his work, Belkin suggested that these three genes could play a role in 2,4-DNT and 2,4,6-TNT metabolism because when these genes were knocked out, the response of the yqjF and ybiJ promoters decreased 1,000 times.[1]

As a consequence, these three genes were used in the development of some of our constructs aiming at enhancing the response of yqjF and ybiJ promoters under the presence of Landmine compounds.

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.