Question

Design a detailed, step-by-step, experiment to a) Detect the presence of Nitrate reductase gene in your unknown bacterial sample b) Detect the presence of Nitrate reductase enzyme in your unknown bacterial sample

Answer 1

Answer:            This question i have to write pointswise,because it's easy to understand you....

a):    Detection of Nitrate Reductase gene in Bacillus subtilis

• Nitrate Reductase is the enzyme which produces nitrites from nitrates, this is an oxygen sensitive synthesised from the Nas DEF gene,
• For the detection of the gene and quantification of gene also can be determined, design two primers and run the PCR by taking desired bacterial Genome , there we can't detect the genes.
• Designing of probes, cDNA probes, Colony hybridization techniques are employed for detection of Nitrate Reductase in the Bacterial cell/Genome.

b): Nitrate Reductase enzyme assay is performed to quantify the amount of enzyme.

• NITRATE reacts with NADH in presence of Nitrate Reductase to produce Nitrite and NAD and H2O, the amount of NAD formed is directly proportional to conversion of Nitrate into nitrite.
• that is directly proportional to Nitrate Reductase enzyme.
• the reduction reaction step depicts the enzyme activity.
• Optical density values are measured and plotted graphsbto determine enzyme activity

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