Nitrogen in its molecular form (N2) is the most abundant and an essential compound in earth’s atmosphere to sustain life, but our environmental interest has always manifested its conversion into other chemically reactive forms. With prime focus on carbon pollution in recent decades, alarming increase in the levels of ‘Reactive Nitrogen’ in the environment has gone largely unnoticed (UNEP Frontiers: The Nitrogen Fix:2018-2019; Emerging issues of environmental concern). The various forms of reactive nitrogen on coming in contact with water are known to form one of the compounds as N-Nitrosamines, which have posed a potential threat to human health during drinking water disinfection. N-Nitrosamines are known to be mutagenic, teratogenic and potent human carcinogens present in varied packed food items, cosmetics, pharmaceuticals, industrial effluents and agricultural wastes etc., and form a major chunk in particulate matter, but there most alarming concentration has been in potable water. UNEP yearbook 2014 states that it’s not the magnitude of problem but the negligible progress made to curb the problem which is a grave concern.
E.coli has a natural adaptive response mechanism known as the Ada response. It is a DNA damage repair mechanism that gets induced by an O6-methylguanine (O6-MeG) lesion formed due to methylation of DNA in the presence of alkylating agents like nitrosamines. This damage caused in DNA due to methylation is repaired using four Ada response proteins, encoded by three different operons of the “ADA Regulon”. These proteins include Ada methyltransferase, AlkA glycosylase, AlkB dioxygenase and AidB protein. The methylated Ada protein acts as a transcriptional activator for the operons involved in the synthesis of these proteins.
The primary goal of our project is to engineer E.coli that detects the presence of alkylating agents in drinking water
For this, we have constitutively expressed the Ada response protein which is released in E.coli upon DNA damage caused due to alkylation. Its constitutive expression allows us to report the slightest concentration of nitrosamines present in water sample. This Ada protein undergoes methylation and further activates the synthesis of the reporter protein present downstream to the ADA-AlkB promoter showing the presence of the targeted compounds via fluorescence
We also propose to specifically degrade NDMA
Pseudomonas mendocina KR1 possesses the T4 monooxygenase gene which has the ability to degrade NDMA. We will introduce this gene in E.coli which, in the presence of toluene, would get activated by methylated Ada protein and help in the degradation of this specific nitrosamine. The completion of this process would be visualized by the change in the colour of the reporter protein.