Ayoung investigator would like to express and purify a newly discovered gene. she decides to clone the gene into a plasmid that contains the sequence for a hexahistidine tag. plasmids that contain a sequence encoding a hexahistidine tag will produce a recombinant protein with an additional six histidine residues attached to the n (or c) terminus. her gene of interest will be expressed as an n-terminal fusion protein containing the six residue histidine tag (n- his6-protein -c). order the steps for construction of the expression plasmid. (note: the gene of interest will be inserted between the noti and hindiii sites.)

step 1:

step 2:

step 3:

step 4:

step 5:

the options are:

-introduce notl and hindlll restriction sites at the ends of the gene of interest via pcr.

-digest the plasmid and dna encoding gene with the notl and hindlll restriction endonucleuses

-transform the plasmid into e. coli for expression

-purify the protein using a column of immobilized ni^2+

-ligate the cut plasmid and dna fragment