Team:IIT Delhi/Parts

After working day and night, We successfully designed and submitted the following parts with the sole aim of reducing environmental pollution and creating the world a better place to live
✔ BioBrick:1 Part:BBa_K1395001-nrfA gene (Nitrite reductase enzyme) under constitutive promoter ✔

✔ This part is a combination of constitutive Promoter (BBA_J23119) and nrfA gene (BBA_k1153001). The nrfA gene (Biobrick no. BBA_k1153001) encodes for the Nitrite reductase enzyme (also known as ccNiR, source E.coli K12) which detoxifies nitrogen oxides (NOx) to ammonia (NH3). This gene was obtained from BBA_k1153001 and is 3A assembly (RFC10) compatible. The Constitutive Promoter (BBA_J23119) is the "consensus" promoter sequence and the strongest member of the constitutive promoter family developed by John Christopher Anderson of UC Berkeley.This promoter can be used to tunes the expression level of constitutively expressed parts.The nrfA gene is expressed under this constitutive promoter. Its expression is based on the availability of RNA polymerase holoenzyme and the expression is not affected by any transcription factors.

✔ Consequently, nrfA may well act on sulphite ions in the cell. Sulphite reduction by NrfA generates sulphide in a six-electron process that appears to parallel nitrite ammonification although the reaction pathway, and indeed the physiological role of this reaction, are presently unclear. Steady-state parameters describing NrfA sulphite reduction that may inform on the possible in cells. Consequences of interactions between sulphite and NrfA have not been reported to date. However, where rates of sulphite reduction are documented they are at least as high as those of dedicated sulphite reductases although several orders of magnitude less than those for nitrite reduction under comparable conditions. It may also be significant that Sulfite (SO32-) can bind as the distal ligand to the active site heme. This suggests that sulphite will compete with nitrite and nitric oxide for binding to NrfA and, since it is reduced considerably more slowly than those substrates, its presence may have a significant impact on the rates of reduction of the nitrogenous substrates.

✔ Biobrick 2: Part:BBa_K1395002 sqr gene (sulfide quinone reductase) under constitutive promoter ✔

✔ This part contains the sqr(sulphide quinone reductase Part:BBa_K896000 ) gene downstream with the constitutive promoter(BBA_J23119). The sqr gene(Part:BBa_K896000) encodes a protein of 427 amino acid residues with a theoretical molecular weight of 47 kDa. The sqr gene is expressed under a constitutive promoter and this enzyme converts the sulphide (S-2) to elemental Sulfur. Its expression is based on the availability of RNA polymerase holoenzyme and the expression is not affected by any transcription factors and is 3A assembly (RFC10) compatible. The Constitutive Promoter (BBA_J23119) is the "consensus" promoter sequence and the strongest member of the constitutive promoter family developed by John Christopher Anderson of UC Berkeley. This promoter can be used to tunes the expression level of constitutively expressed parts.

This part codes for the protein sulfide quinone reductase which is a FAD dependent oxidoreductase. Sulfide-quinone reductase (SQR),an ancient flavoprotein, is obligatory for growth on sulfide as hydrogen donor in photo and chemolithoautotrophic bacteria. It is a unique enzyme which is responsible for transfer of electrons from sulfide into the quinone pool. This enzyme converts the sulphide to Sulfur by the reaction.

✔ Biobrick 3: Part:BBa_K1395003 sqr gene (sulfide quinone reductase) under constitutive promoter ✔

This part is composite biobrick having parts of biobricks Bba_K896000 and Bba_K896001. The first part is sqr gene obtained from biobrick Bba_K896000. In this part the gene is expressed under a constitutive promoter and this enzyme converts the sulphide (S-2 ) to elemental Sulfur. And the second part is cysI gene obtained from Bba_K896001 which converts sulphite(SO32-) to sulphide (S-2 ) . So, these two parts codes for proteins which simultaneously work to convert sulphite (SO32-)to elemental Sulfur.

Testing of Biobricks
Expression of protein: The plasmids of positive clones were digested by EcoR1 and Pst1I to check the release of insert and clones were confirmed. The positive clone was sequenced by Chromus Biotech and sequence was confirmed by Clustal W sequence alignment program. The E. coli cells containing our clones were grown in LB medium supplemented with 34 µg mL-1 chloramphenicol as selective agent at 37ºC till OD600 reaches 1.0. The expression of proteins was checked on SDS-PAGE.

Activity Assay of nrfA:
For activity assay cells were sonicated for 10 minutes at 50 amplitude with 5 second pulse on and 5 second pulse off. After this the cells were centrifuged at 10000 rpm for 20 minutes at 4C to separate the cell lysate. In activity assay 50mM Sodium Phosphate Buffer (pH – 7.2), 2mM KCN, 0.16mM DCPIP, 10mM tyramine was used to test the activity of enzyme. DCPIP used as a redox dye. Oxidized, DCPIP is blue with a maximal absorption at 600 nm; when reduced, DCPIP is colorless.