Part:BBa_K1866003

Promoter+RibosomeBindingSite+Sqr gene (in pSB1A3)

This part consists of the Sulfide Quinone Reductase Sqr gene under the control of a strong promoter with a Ribosome Binding Site. The Sqr gene catalyses the conversion of hydrogen Sulfide gas to elemental Sulfur.

Figure1: Structure of the Sulfide Quinone Reductase complex. Courtesy: PDB

The Enzyme:

Sqr gene encodes for the Sulfide Quinone Reductase protein. This enzyme converts Hydrogen Sulfide gas to elemental sulfur, which is released as granules by the host. This enzyme plays an important role in the electron transport system of the host.

Figure2: Schematic representation of the reaction catalyzed by Sulfide Quinone Reductase.

The engineered bacterial system:

Team IIT Delhi has designed a bacterial system that overexpresses the Sulfide Quinone Reductase enzyme. We cloned the Sqr 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 Hydrogen Sulfide, an irritable, toxic and corrosive gas.

Biology of the engineered system:

The enzyme Sulfide Quinone Reductase has been found in many prokaryotes like Cyanococcus spp. that respire anaerobically in environments rich in Sulfate. Sulfates are transported into the cell, which are then converted into Sulfides. These Sulfides are then converted into Sulfure that is released by the host, with the concomitant transfer of electrons. Thus, the activity of Sulfide Quinone Reductase is one part of the big pathway of conversion of Sulfates to Sulfur. The engineered bacterial system would catalyze the final step in the pathway thereby reducing the levels of Hydrogen Sulfide in the feed.

Gene construction & cloning:

The general cloning strategy for NrfA consists of the following steps.

1. The biobrick BBa_K896000 (Sqr), previously submitted by NYMU iGEM 2012 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. (Sqr) was doubly digested by XbaI and PstI to yield Xba-(Sqr)-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)-(Sqr)-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:
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

It is evident from the picture that we were not successful in obtaining expression of Sulfide Quinone Reductase protein.

The team is currently troubleshooting the issues and is planning to proceed with working on Sqr.

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]