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. Ranging from nanograms to micrograms; detection of these N-nitrosamines in water supplies and their subsequent removal has been a raging environmental and public health concern. Some analytical methods currently in wide usage - a combination of chromatography (HPLC/LC/GC) and mass spectrometry are inadequate for detecting N-nitrosamines at low ng/L concentrations in water and are highly expensive requiring well equipped laboratories.
One of the properties of nitrosamines is their potential to alkylate DNA. We propose to make an E.coli based biosensing solution to detect alkylating agents like nitrosamines. On the same tone, we also propose a cell based biosensing solution that can not only detect but degrade one of the member of these alkylating agents i.e. NDMA. For this, we have designed to Biobricks, namely:
General detection Biobrick.
Construct 1: General Detection Biobrick
This biobrick engineers E.coli such that it specifically detects the presence of alkylating agents like nitrosamines in drinking water.
Mechanistically, Nitrosamines, being alkylating agents, alkylate the DNA by forming O6-MeG or O4-MeG lesion. Ada molecule, a natural DNA repairing chemosensor in E.coli, due to its methyltransferase activity, repairs the methylated bases. It so happens, when Ada comes in contact with O6-MeG or O4-MeG lesion formed by the alkylating agents like nitrosamines. The methyl-shift reaction that takes place between Ada and methylated lesion is irreversible, thus suicidal. But, this methyl-shift leads to a hype in binding affinity of Ada with DNA and consequently acts as an efficient transcriptional activator of three operons involved in the ADA regulon (The regulon for DNA repair). We have used Ada-induced Ada-AlkB promoter which is one of the promoter in the regulon. This promoter is naturally succeeded by ADA-AlkB gene but we are replacing this gene with Reporter sequence, drawn from iGEM repository, to detect the presence alkylating agents.
Undermentioned is the detailed description of parts that were used in the biobrick:
It is a constitutive promoter sequence which is to be used for the continuous expression of ADA-AlkB operon. It is one of the three operons of the Ada regulon. The constitutive promoter is required to enhance the expression of ada protein which is otherwise quite low in the E.coli cell. (Uphoff et al, 2016)
This ribosomal binding site is a part of the Anderson RBS family that is compatible with E. coli and other prokaryotes for general protein expression.
ADA-AlkB gene optimized sequence (Registered as BBa_K3587000):
It is the optimized gene sequence of one of the three operons of the ADA Regulon. It encodes for the ada protein which (when methylated as part of ADA response) acts as a transcriptional activator for the Ada-AlkB promoter. This sequence used in the biobrick is optimized in order to attune itself with the prefix and suffixed to be used .
It is a double terminator consisting of BBa_B0010 and BBa_B0012 which is designed by Reshma Shetty. It is one of the most reliable terminator in iGEM repository and is frequently used.
ADA-AlkB Promoter + RBS (Registered as BBa_K3587005):
It is an efficient promoter of Ada regulon with a Ribosomal binding site (RBS). This promoter exhibits about a 10-fold higher affinity for the ada protein (Landini & Volkert, 1995) than the other promoters of the regulon. The reporter sequence is placed downstream to this promoter.
This GFP sequence from IGEM repository is one of the frequently used reporter gene.
E. coli possesses Ada protein at a low concentration, usually 0 to 6 molecules per cell (Uphoff et al, 2016). The Ada protein is made to express constitutively, to report the slightest concentration of nitrosamines present in water sample quickly. 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.
Construct 2: Pro Biobrick
The specificity of our biosensing solution lies in the Pro biobrick which is made up of detection biobrick and some added sequences. The addends include the Ada alkB promoter placed upstream of a T4-MO gene followed by a reporter and a stop codon. Due to the pandemic, our group was not able to work further on this biobrick.
Our further working on this gene will help us to accomplish a biosensing solution that can detect the presence of alkylating agents like nitrosamine in water and selectively degrade the hazardous NDMA. Every aspect of its working will be characterized with the emission of a different reporter.