From our analysis of possible fluorescent protein reporters, we selected yellow fluorescent protein (YFP) and red fluorescent protein (RFP)
2. Construction: Overlap PCR
We first synthesized the estrogen sensor by cloning the estrogen responsive intein into the T7 RNA Polymerase. The intein was inserted between the 491 and 492 residues of the T7 RNA Polymerase using overlap PCR. We did this by first using PCR to piece the N-terminus of the T7 RNA Polymerase to the N-terminus of the ''S. cerevisiae'' VMA intein. Another PCR reaction was set up to piece together the C-terminus of the intein and the T7 RNA Polymerase. The third reaction pieced together these two parts, along with the estrogen ligand binding domain to produce the intein sensor, which would splice in the presence of estrogen to produce a functional T7 RNA Polymerase. We then checked to see that we made the desired product by running this through a 1 % agarose gel and looking for the specific size bands. In order to create the second plasmid indicating whether the intein was spliced in the presence of estrogen, we ligated the T7 promoter and RFP into the pSB3K3 plasmid. This plasmid, along with the plasmid containing the estrogen sensitive intein, were co-transformed into ''E. coli'' MACH cells to be tested with estrogen
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