Promoter+RibosomeBindingSite+NrfA (in pSB1C3)
The assimilatory Nitrite Reductase gene under the control of a strong Promoter Anderson J23100 with a Ribosome Binding Site was cloned onto the pSB1C3 plasmid backbone. This gene catalyses the conversion of Nitrite (NO2 -) to Ammonium (NH4 +).
Figure1: Structure of the Assimilatory Nitrite Reductase complex. Courtesy: PDB
The entire cassette can be excised from the backbone and used as per requirement.
NrfA gene encodes for the assimilatory Nitrite reductase protein. This enzyme, localized in the periplasmic space of the bacterium, catalyzes the conversion of Nitrite to ammonia, which can be assimilated by the host into building blocks like proteins. Also, another important product of the reaction, NAD(P)+ act as reducing equivalents that are an inherent part of the electron transport system in the host.
Figure2: Schematic representation of the reaction catalyzed by Assimilatory Nitrite Reductase.
The engineered bacterial system:
Team IIT Delhi has designed a bacterial system that overexpresses the assimilatory Nitrite Reductase enzyme. We cloned the NrfA gene encoding the enzyme onto an expression vector pSB1C3 under the control of a strong promoter. The recombinant DNA vector can be stably maintained in the host under antibiotic selection pressure. This engineered bacterium is capable of reducing the levels of Nitric Oxide, a potent greenhouse gas, which is also an intermediate in the Nitrite-to-Ammonia conversion.
Biology of the engineered system:
The enzyme Nitrite Reductase has been found in many prokaryotes like Pseudomonas aeruginosa that respire anaerobically in environments rich in Nitrates. Nitrates are transported by Nitrate transporters into the cell, which are then converted into Nitrite by Nitrate Reductase enzyme. These nitrites are then converted into Ammonia that is assimilated by the host, with the concomitant regeneration of the reducing equivalents. Thus, the activity of assimilatory Nitrite Reductase is one part of the big pathway of conversion of Nitrates to Ammonia. The engineered bacterial system would catalyze the final step in the pathway thereby reducing the levels of Nitrite in the feed.
Gene construction & cloning:
The general cloning strategy for NrfA consists of the following steps.
Confirmation of the clone:
We performed a double digestion by EcoRI and PstI to release the cloned fragment from the recombinant vector. The digestion products were resolved on an Agarose Gel by Electrophoresis and the size of the fragment confirmed the correct clone.
Figure3: Clone confirmation
Characterization of the construct:
To check the expression of the gene, we analyzed the proteome on an SDS-PAGE. The profile that was obtained is given below:
Figure4: SDS PAGE to check expression
Functional assay of Assimilatory Nitrite Reductase (Concept proof):
Practical application of Nitrite Reductase- The Prototype <p>The real-life problem that we thought of addressing was the reduction of the levels of harmful greenhouse gases like Nitrogen oxides that are usually found in the exhaust fumes of Diesel-based engines.
We designed a prototype model to address the issue. A schematic representation of the model is given below.
Figure5: Schematic representation of the Prototype
To test the effectiveness of our prototype, we used the exhaust of a diesel generator as a feed to the system and analyzed the outlet for the presence of the Nesseler-Ammonium precipitate based on the idea that the quantity of the precipitate would be proportional to the quantity of the reactant (Nitric Oxide) in the feed.
Feed processing: The feed had to be processed before passing to the engineered bacterial system. It consisted of following stages.
1. Minimal Media Growth:
This experiment failed, even though we saw a pellet after centrifugation following growth. However, we later realised that the pellet was not of our cells, but of ampicillin, which had formed some sort of white precipitate with the media. Hence, this resulted in a failure.
2. pH monitoring:
The experiment showed results as expected, with the pH of our clones being higher than the control, which can be attributed to the presence of excess ammonia in the solution. We also saw that at concentrations of Nitrite greater than 2mM, the pH showed a sharp decline. This could be due to the fact that high concentrations of nitrite (NO¬2-) become toxic for the cells, due to which cell death occurs.
3. Indophenol test:
a. Monitoring with time- This experiment was successful, as it showed an increase of OD values with increase in time, before finally saturating. The OD values were more than that of DH5α cultures throughout, pointing to the fact that more ammonia was being formed in our clone cultures.
b. Monitoring with different nitrite concentrations- This experiment was also successful, with OD values showing a trend similar to the one above. At high concentrations of Sodium nitrite, the OD values showed a sharp decline, which can again be attributed to high concentrations of nitrite (>2mM) being toxic to the cells.
4. Nessler’s test (LB growth):
This experiment was also successful, showing trends as expected. At high nitrite concentrations, the pellet size reduced drastically (the reason being toxicity at high nitrite concentration).
Tests were carried out on the prototype, taking in exhaust from a diesel burner and passing it through the system. The desooter tank worked even better than expected, collecting soot out from the exhaust (which was weighed after each run). NOx sensors placed at the edge of the silica gel tube confirmed that the gel was catalysing the reaction that we intended to do, converting NO2 to NO.
<p>We could, thus, successfully confirm the reduction in the levels of Nitrogen Dioxide (NO2) in the exhaust fumes of an actual diesel generator. The prototype can be optimized further for increasing the efficiency of the system.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 380
- 1000COMPATIBLE WITH RFC