"
Team:Carnegie Mellon/Sensor
From 2014.igem.org
The Sensor
Fluorescent Protein Analysis
From our analysis of possible fluorescent protein reporters, we selected yellow fluorescent protein (YFP) and red fluorescent protein (RFP)
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
Results
While we were able to successfully construct our sensor using overlap PCR, and a double transformation into E. coli MACH cells (a W strain), we were unable to get a significant fluorescent signal from these cells in the presence of estrogen. The E.coli cells containing the sensor were tested with 1 - 20 ul of 10 mg/ml solution of estrogen in ethanol. We grew the cells in the presence of estrogen at 30 °C for 4 hours, taking samples every 30 to 60 minutes. Our model also predicted that we would not be able to detect any fluorescent signal.
