Composite

Part:BBa_K1866010

Designed by: Stefan Oehler   Group: iGEM15_IIT_Delhi   (2015-09-18)

Promoter+RibosomeBindingSite+NosZ+SuperYellowFluorescenceProtein (in pSB1C3)

This part consists of the gene NosZ that encodes for an enzyme named Dissimilatory Nitrous Oxide Reductase.

This enzyme catalyses the conversion of Nitrous Oxide, a greenhouse gas to Nitrogen gas. The NosZ gene is cloned under the control of a strong Promoter with a Ribosome Binding Site for optimum expression.

This part also consists of a Super Yellow Fluorescence Protein tag to check the expression of the NosZ gene.

Figure1: Structure of the Nitrous Oxide Reductase complex. Courtesy: PDB

NosZ_protein_structure_IIT_D.jpg

The Enzyme:

NosZ gene encodes for the dissimilatory Nitrous Oxide reductase protein. This enzyme, localized in the periplasmic space of the bacterium, catalyzes the conversion of Nitrous Oxide to molecular Nitrogen gas, which is then released by the host into the atmosphere. Thus, denitrification, which is an ecologically important step, is catalyzed by this enzyme.

The engineered bacterial system:

Team IIT Delhi has designed a bacterial system that overexpresses the dissimilatory Nitrous Oxide Reductase enzyme. We cloned the NosZ 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 Nitrous Oxide, a potent greenhouse gas, by a process known as denitrifiication.

Biology of the engineered system:

The enzyme Nitrous Oxide Reductase has been found in many prokaryotes like Pseudomonas stutzeri that respire anaerobically in environments rich in Nitrates. Nitrates are transported by Nitrate transporters into the cell, which are then converted eventually into Nitrous Oxide by the enzymes of the denitrification pathway. This is then converted into Nitrogen gas that is released into the atmosphere.
Thus, the activity of dissimilatory Nitrous Oxide Reductase is one part of the big pathway of conversion of Nitrates to Nitrogen gas. The engineered bacterial system would catalyze the final step in the pathway thereby reducing the levels of Nitrous Oxide in the feed.

Gene construction & cloning:

The general cloning strategy for P+RBS+NosZ+SYFP consists of the following steps.

  • 1. The biobrick BBa_K1866001 (NosZ+SYFP) was prepared by Team IIT Delhi iGEM 2015.
  • 2. Another biobrick consisting of the Promoter and Ribosome Binding Site BB_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. (NosZ+SYFP) was doubly digested by EcoRI and XbaI to yield EcoRI-(NosZ+SYFP)-XbaI construct.
  • 5. Ligation was set up to obtain the desired construct as shown below.

    EcoRI-XbaI-(P+RBS)-(NosZ+SYFP)-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.

    Figure2: Clone confirmation.

    Clone_confirmation_IIT_D_2015.jpg


    Characterization:


    To check the expression of the gene, we analyzed the proteome on an SDS-PAGE. The profile that was obtained is given below:

    Figure3: SDS PAGE to check expression

    SDS_PAGE_IIT_Delhi.jpg



    Functional assay of Assimilatory Nitrite Reductase (Concept proof)


    Product Estimation:
    The Nitrite that was passed in the feed was converted to ammonia by the Nitrite Reductase. The spent medium (without the biomass fraction) was incubated with Nesseler’s reagent as previously described. The precipitate that was obtained was quantified for its mass after centrifuging and then removing the supernatant. The amount of the precipitate was in direct proportion to the quantity of Nitrite, and thus, Nitrous Oxide in the exhaust fumes of the diesel generator.


    Practical application of Nitrite Reductase- The Prototype

    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.

    SCHEMATIC HERE.

    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:

    We could, thus, successfully confirm the reduction in the levels of Nitrous Oxides 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]


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