Question

You are studying a mouse gene named MtoA. You know that the protein product of this gene localizes to mitochondria in mice. Your goal is that you want to know whether mouse MtoA protein is capable of localizing to mitochondria if produced in yeast cells. You decide to clone a gene into a translational fusion vector that will add jellyfish DNA sequence encoding the GFP proteins (238 amino acids) in place of the normal stop codon. Unfortunately, you can't find any restirction enzyme recognition sequences in the MtoA gene that would allow you to clone your DNA into the multiple cloning stie.  

a) What method can you use to amplify the gene?

b) Briefly describe a variation to this method that would help solve you problem.

c)You make the plasmids in a test tube, then transform them into E. coli. You grow the E. coli and extract the plasmid. What 2 methods could you use to determine if the plasmid has the MtoA-GFP fusion?

d)Must the plasmid contain a promoter that works in jellyfish? If yes, state which specific gene on the plasmid will be controlled by the promoter?

e)Must the plasmid contain a promoter that works in E. Coli? If yes, state which specific gene on the plasmid will be controlled by the promoter?

After confirming that you plasmid is correct, you transform it into yeast. You analyze transformed colonies by Northern and Western blotting. As a control you also have cloned into yeast a non-GFP version where your gene retains its stop codon in the normal position and does not have the GFP sequence. Draw bands in these two blots with the expected results.

Answer 1

a) use a PCR reaction

b) use primers having sequence fir restriction enzymes upstream of the complementary sequence.

c)(i) design specific primers and run a pcr.

(,ii) observe the transformed E. coli cells for the presence of green fluorescence under an fluorescence microscope.

d) no, a jellyfish promoter is not required as we do not need this fusion protein to be expressed in jelly fish.

e) yes, an E. coli specific promoter is required as we need the fusion protein to be expressed in E. coli. mtoA-gfp needs to be placed under this promoter.