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Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication-transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.

Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.