This experiment is designed to let you explore the techniques for expression of a recombinant protein, its purification and detection in bacterial cells. You should develop questions that you want to answer.
For this experiment you will be given a plasmid, pcDNA3.1His6LacZSD, which contains the gene for b-galactosidase. This plasmid is designed to express this gene in mammalian COS-7 cells from the CMV promoter and has been modified from it's original for to also express transcripts from the T7 promoter in E. coli JM109DE3 or BL-21cells. There is a region in the fused leader that contains a series of 6 Histidines in a row (His6). This motif is useful during purification because it will bind Ni-NTA agarose and allow all non-His6 containing proteins to be washed off the resin. The His6 containing proteins can then be eluted off the resin with high levels of imidazole.
The plasmid sequence can be downloaded here: pcDNA3.1His6LacZSD