A large biotechnology has subcontracted your services to pro

A large biotechnology has subcontracted your services to produce human lactate dehydrogenase. You decide to use DNA extracted from human fibroblasts to insert the gene into a bacterial plasmid. The lactate dehydrogenase gene has a length of 20417 base pairs. There are several introns in the gene. The exons are as follows Base pairs that form exons: Moreover, on this sequence, the open reading frame spans from the position 7409 to 17847 on the DNA. What is the length of the messenger RNA (excluding the poly(A) tail)? What is the length of the polypeptide chain (number of amino acids prior to any posttranslational modifications)? You decide to produce your enzyme in E. coli using recombinant DNA. To facilitate the purification of the enzyme, you add a 5\' signal peptide to the cloned sequence to allow the secretion of the protein. You measure the enzyme activity both in the medium and in lysed cells and you notice that there is almost no secreted enzyme. What is the most likely cause for the lack of secretion of the enzyme into the medium? You observe a contamination in your medium. At the same time, you notice that the pH of the medium is much lower than usual. Based on the metabolism of different microorganisms, explain what metabolic pathways and molecules may cause this decrease in pH.

Solution

1.

a. The length of the mRNA sequence is approximately 999 bps

b. The length of the polypeptide chain would be 333 amino acids

2. There might be two reasons for the lack of the enzyme in the medium

a. The signal sequence might have been cleaved.

b. There could have been a mutation in the signal sequence.

3. The change in the pH is due to the carbohydrate fermentation. The contaminants in the media utilise the sugars and anaerobically ferment producing alcohol which lowers the pH of the media. To detect the fermentation of carbohydrates by change of the medium from an orange-red to a yellow colour.