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
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.
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
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
- 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