Please help me, I am deeply confused in this class and trying so hard to underst

Question

Please help me, I am deeply confused in this class and trying so hard to understand. Please give detailed answers. I know some of these might be confusing but your help will really help me learn. The teacher requires very very detailed answers and he gives nearly no (and I mean literally nearly zero) partial credit so I'm dying here. Thank you sooooooo much, you have no idea.

Also, I am nearly leaglly blind and I am dyslexic so if you could please write clear it would help so much. I just want to learn like everyone else. SUPER SUPER THANK YOU.

QUESTION 3 (10 PTS) You work at a biotechnology company that is trying to cure sickle cell anemia. The first step in your research is to take the normal version of the hemoglobin gene and insert it into E. coli. The goal is to get the bacteria to make normal hemoglobin protein for you to use in your experiments. 3A. You attempt this by amplifying the hemoglobin gene from human DNA and inserting into E coli. Your PCR primers amplify the entire gene, including the promoter, ORF, and terminator. You insert this sequence into the E coli genome, but you can't detect any hemoglobin mRNA. Why? Describe a specific change you could make to the gene sequence you amplified that might fix this problem and then explain why this would work. (3pt) 3B. You have a similar problem getting translation to occur, but eventually are able to alter the hemoglobin gene enough to get the E. coli to make the hemoglobin protein. When you purify the protein from the bacteria, however, you find that it is longer than expected. When you look at the order of amino acids, the first 80 amino acids match the expected sequence but then the sequence drastically diverges from the known hemoglobin amino acid sequence. How can you explain this? (2pt) 3C. You are able to fix this problem as well and finally purify hemoglobin protein from the bacteria. But the hemoglobin protein is too short! You go back and analyze your original PCR reaction and find that 1 out of every 4 products in your reaction has the same C>A mutation How could this mutation yield a shortened protein? Use specific nucleotide sequences in your explanation. (2pts) 3D. You guess that this mutation arose from a polymerase error during the PCR reaction. Based on the fact that 1 of 4 products has the mutation, you can say with good certainty which round of amplification this mutation occurred in. How do you know when the mutation arose? It's probably easiest to draw a cartoon. (3pts)

Explanation / Answer

Ans.

3 A.

We can't detect any mRNA which means there is no transcription. this is because the sequence is not inserted downstream to the bacterial promoter and bacterial transcription factors cannot recognize the promoter site and transcription fails to occur.

To fix this problem the amplified foreign DNA can be linked to a bacterial promoter which would ensure the transcription of that gene.

3 B.

The reason for the protein expressed in bacteria being longer than the normal protein is no splicing of introns in bacteria which usually occurs in eukaryotes at mRNA level. introns are stretches of nucleotides that are removed from mRNA prior to translation. however this does not occur in bacteria and introns are retained. that's why after first 80 amino acids the amino acid sequence changes drastically.

3 C.

The C to A mutation can turn a amino acid coding codon (UCA for example which codes for serine) to stop codon (UAA) and hence translation will stop early resulting in a truncated product.

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