The second sensing system we have designed relies on transcriptional repressors. By creating a synthetic repressor that gets cleaved by a specific protease, the extract we create will be sensitive to the protease. This can be used to detect breast and colorectal cancer biomarkers such as MMP-2 and MMP-9 in patients' urine.(Coticchia 2011) This project uses several concepts to sense proteases. First of all, this system relies on a two-hybrid repressor previously described in some detail. (Di Lallo 2001) Secondly, instead of using the two-hybrid as a way of detecting the interaction between two proteins, we created fusion proteins that contain both parts of the two-hybrid repressor. This allowed us to insert a linker sensitive to specific proteases, which would then inactivate the repressor, and allow transcription of the reporter to occur, as shown in the image below.
Project State
Currently, we have all the necessary plasmids in NiCo21(DE3) 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. As we obtain results after the Jamboree, this website will be updated.
