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Team:Carnegie Mellon/Sensor
From 2014.igem.org
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<p>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 <i>S. cerevisiae</i> 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 <i>E. coli</i> MACH cells and Top10 cells to be tested with estrogen</p> | <p>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 <i>S. cerevisiae</i> 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 <i>E. coli</i> MACH cells and Top10 cells to be tested with estrogen</p> | ||
<p><h4>Testing</h4></p> | <p><h4>Testing</h4></p> | ||
| - | <p>The cells containing the sensor were grown overnight at 37 °C. They were then diluted ten fold in LB, and grown at 30 °C and treated with 17-& | + | <p>The cells containing the sensor were grown overnight at 37 °C. They were then diluted ten fold in LB, and grown at 30 °C and treated with 17-&BetaEstradiol from Acros Organics. The <i>E.coli</i> cells containing the sensor were tested with 1, 2 and 10 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. We also tested growth of the cells over time at different temperatures (30 °C and 37 °C). |
<hr> | <hr> | ||
Revision as of 03:22, 17 October 2014
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 and Top10 cells to be tested with estrogen
Testing
The cells containing the sensor were grown overnight at 37 °C. They were then diluted ten fold in LB, and grown at 30 °C and treated with 17-&BetaEstradiol from Acros Organics. The E.coli cells containing the sensor were tested with 1, 2 and 10 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. We also tested growth of the cells over time at different temperatures (30 °C and 37 °C).
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) and Top10 cells, we were unable to get a significant fluorescent signal from these cells in the presence of estrogen. Our model also predicted that we would not be able to detect any fluorescent signal. However, we were able to observe growth of the E.coli cells containing the sensor at 30 °C and 37 °C. In addition, we observed significantly higher fluorescence of the YFP and RFP in their respective controls.
