Team:Pitt/Estrogen/Project

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Project Overview Estrogen Sensor Protease Sensor Three-Hybrid Versatile Sensor Amplification and Quenching

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Estrogen Sensor

Project Background

While it has been shown that transcription in cell-free extracts can rely on RNA polymerases sensitive to small molecules (Pardee 2014), our team decided to test the viability of using such polymerases in our cheap, home-made sensor extracts, rather than in expensive, commercially available extracts. In doing so, we used a part from CMU's iGEM team, the estrogen-sensitive T7 RNA polymerase. This subproject was the first and simplest application of our sensor extracts. Since CMU's iGEM team had been working on an estrogen-sensitive mutant of T7 RNA Polymerase, we decided to use the construct in a cell-free context. Sensors that detect estrogen quickly could be used in a variety of contexts, including but not limited to quantifying estrogen in blood, and detecting estrogen-contaminated water.

The sensor would work roughly as shown above. In the absence of estrogen, the mutant T7 RNAP would be in the incorrect conformation, which would not allow it to transcribe the reporter construct. When estrogen becomes available, the protein folds into its active conformation, which allows it to transcribe the reporter, which eventually produces a visible result. To learn more about the function of the mutant T7 RNAP, visit the CMU team's webpage. Since this project relies on a modified T7 RNAP, we searched for a reporter construct based on a T7 promoter. One of the constructs we found was PT7-GFP-TAG-RFP, which we characterized, and to which we added a contribution. We also constructed a pT7-eGFP part, which is our best characterized part.

Project State

Currently, we have all the necessary plasmids in BL21 cells, which are ideal for this project. We have created sensor extracts using these cells; however, we have run into problems involving dead enzymes which have delayed our results. Results typical of our extracts are shown below:

As we obtain results after the Jamboree, this website will be updated.