Question

You want to make E. coli bacteria that glow green. Your plan is to insert the gene for green fluorescent protein (GFP) from a jellyfish (a eukaryote) into the genome of the bacteria so that the mRNA is transcribed by the E. coli cells and translated into protein. What changes do you need to make to the jellyfish GFP gene sequence to make sure that the bacteria will make a functional protein? Why? (1 point)

A type of amatoxin binds to the carboxy-terminal domain of RNA polymerase II and renders the CTD completely non-functional. Obviously this would be disastrous; describe two things that would be affected in a cell poisoned with this amatoxin and briefly explain your answers. (1 point)

Draw a eukaryotic gene with two exons and one intron. Label all the components of the gene, including the 5’ and 3’ ends and indicating the region of transcribed sequence. (1 point)

Now draw what both the pre-mRNA and mature mRNA would look like from the same gene. Label all important features of these transcripts. (2 points)

Answer 1

Hi,

The jellyfish is an eukaryote and e.coli is a prokaryote. The jellyfish GFP gene has introns and Exxon’s , whereas the prokaryotes do not contain any introns. Also the jellyfish GFP promoter is eukaryotic which will not work in prokaryotic. Before cloning the gene in e.coli, the introns need to be removed and promoter needs to be changed. Best way is to use a mRNA of gGFP and reverse transcribe it and clone it using pBAD vector.

2. The CTD of pol II is necessary for promoter recognition and binding. It has is necessary for successful initiation off transcription. Under the effect of toxins the RNA pol would find loosely to promoter. It will also result in abortive initiation.