Part:BBa_K1866006

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.

The Enzyme:

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.

1. The biobrick BBa_K1153001 (NrfA), previously submitted by KENT iGEM 2013 team, was obtained from iGEM repository.

2. Another biobrick consisting of the Promoter and Ribosome Binding Site BBa_K880005 (P+RBS) was also procured.

3. (P+RBS) was doubly digested by EcoRI and SpeI enzymes to yield Eco-(P+RBS)-Spe construct.

4. (NrfA) was doubly digested by XbaI and PstI to yield Xba-(NrfA)-Pst construct.

5. A linearized vector was prepared by double-digestion with EcoRI and PstI.

6. The 3A assembly was performed to clone the desired construct as shown below into the linearized backbone.

EcoRI-XbaI-(P+RBS)-(NrfA)-SpeI-PstI

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):

Product Estimation:

1. Minimal media growth- Following a research paper by Clarke et al., a culture of our cloned E.coli DH5α culture was grown on a minimal media containing 5% Luria Broth (by volume), 40mM Potassium Nitrate, 20mM Fumarate and 0.4% glycerol for 16 hours, in anaerobic conditions. The cells were then pelleted by centrifugation and were then to be sub cultured. 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 (LB Growth)- Cultures of our clones were grown anaerobically in 5ml of luria broth, along with standard DH5α cells, used as control. These were then subcultured into 50 ml cultures, also grown anaerobically, with different concentrations of Sodium Nitrite. After 16 hours of growth, the cultures were taken out and the pH of the solution was taken for all different concentrations of Nitrite.

3. Indophenol Reagent test: The outlet stream was treated with the Phenol solution (10% Phenol w/v solution in 95% Ethanol) and Sodium Nitroprusside solution (0.5% w/v in deionized water) sequentially. Then the content was treated with an Oxidizing solution of 100ml of Alkaline solution (20% Trisodium citrate and 1% Sodium Hydroxide w/v) and 25ml Sodium Hypochlorite. The absorbance was measured at 640nm after 1 hour incubation.

a. Monitoring with time- For a concentration of 1mM Nitrite, multiple cultures were inoculated. After regular intervals of time, the indophenol test was carried out, and OD540 values were noted.

b. Monitoring with different nitrite concentrations- 50 ml Cultures were inoculated with different concentrations of Sodium nitrite. After 16 hours of growth, the indophenol test was carried out, and OD Values were taken.

4. Nesseler's Reagent test: Cultures of our clones were grown anaerobically in 5ml of luria broth, along with standard DH5α cells, used as control. These were then subcultured into 50 ml cultures, also grown anaerobically, with different concentrations of Sodium Nitrite. After 16 hours of growth, the cultures were taken out and nessler’s reagent was added to a 1ml aliquot. Nessler’s reagent forms a reddish brown precipitate on reacting with ammonia, and is extremely sensitive. This culture was then centrifuged at maximum speed (13,400 rpm) for 10 minutes, and the media was decanted and dried out, leaving only the red ammonia pellet. This was then weighed against DH5α culture (given the same treatment), using the DH5α treated sample as blank (mass of ammonia pellet taken = 0).

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. Removal of insolubles.

2. Silica Gel-based catalysis for conversion of Nitrates in the exhaust to Nitric Oxide.

3. Bubbling Nitric Oxide-containing feed through the medium containing the engineered bacterium to yield Nitrite.

Results:

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).

Prototype Results:

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 380
  
• 1000
  COMPATIBLE WITH RFC[1000]