Question

1. You have cloned the gene sequence of the novel eukaryotic protein you have named Protein Bob (2,100 bpin length). You have used PCR to amplify up the Protein Bob gene from your isolated genomic DNA. After growing up colonies and testing the plasmid you discovered that your cloning protocol has gone according to plan and you did every step correctly, there was no human error. Yet, there is no functional protein production. Explain the reasoning behind this problem. How would you rectify the problem?

Answer 1

Answer. There can be several possiblities of not getting a functional protein.

1) Eukaryotic genomic DNA contains introns in it. During the cloning of the gene these sequences will definitely be in the clone. A bacteria does not have introns so as no intron removal machinary. If the expression system is a bacteria then this clone can only synthesize mRNA but no further processising of that mRNA and thus no functional protein.

2) The PCR fragment to be cloned must have all the regulatory sequences for protein and mRNA synthesis. These sequence lies upward from the gene. So if the pretein synthesis starts from the nucluotide AUG and ends at UAG and this total is 2100bp. That means you have cloned the coding region only. But you also need to add upstream and downstream sequences like 5'UTR, RBS and all.