After your graduation you are employed as a Molecular Biologist at Palapye Biote
ID: 196392 • Letter: A
Question
After your graduation you are employed as a Molecular Biologist at Palapye Biotech Company owned by a prominent businessman with a sketchy view of molecular biology techniques. The company produces Lysine for a duet of industrial applications. Lysine amino acid residues are polymerized in the formulation of a novel antibiotic supplement. Single lysine residues are sold as a supplement for cattle feeds, for a thriving Botswana beef export industry. However, the bacterium, Escherichia gaboronensis produces a limited amount of the amino acids to serve the market. An economist who studied at one of the best institution in the country advices you that an increase as small as 1 % in the production of the amino acid, will earn the company BWP 15 000 000, 00 in a year. Among your investigations you find another bacterium, Escherichia palapyensis that has a better promise to produce elevated levels of the amino acid. Your advisor then recommends that you investigate the operon involved in the biosynthesis of the amino acid in the newly found bacterium. You sleep over it and then decide to alter the production media by adding amino acids and then investigate how the operon expression is affected. After performing a Lys operon mRNA Northern blot and Western blot of the two other proteins expressed from the same mRNA, LysD and LysE, your results are as follows;
Briefly describe the methodology used to obtain these results.
Amino acids Amino acids Amino acids Northern blot of Lys Operon Western blot of Lys D Western blot of Lys EExplanation / Answer
Hi,
The methodology to obtain the given results:
1. Northern blot: It is a technique to detect the expression of mRNA in given cell samples. The RNA is isoalted and separated into single strands. These are then separated according to their sizes using gel electrophoresis. After separation, the RNA fragments are transferred to a blotting membrane.Afte the blotting, the probe which has been specifically designed for LysD and LysE mRNA is flushed on the blot. These probes will bind to the specific mRNA. This hybridization can be visualised using radiolabelling or fluorescent molecules.
2. Western blot : This technique is used to identify and proteins in the given tissue sample. The proteins are isolated and separated using PAGE. The separated fragements are transfered to a blotting membrane (Nylon sheet). All the proteins get transfered to the blot. Now specific antibodies designed to bind to the LysE and LysD proteins are synthesized. These antibodies bind to the proteins and also to an enzyme conjugate. Afte the binding, the membrane is flushed with a substrte for enzyme to act upon. After enzyme reaction, the substrate is converted into a product which can be colorimetric o fluorescent. Thus the presence of protein can be identiied. Most commonly used enzyme conjugates are horseradish peroxidase (HRP) or alkaline phosphatase (AP).