One Step at a Time

      Having finished our tech spec review with a pretty successful presentation, our group has decided to move forward with our cancer detection project.  These last few weeks have been largely focused on ways to improve the project and make it more specific.  We started out by identifying several problems we needed to address - we needed to make sure our system could detect cancer "early" (with as few cancerous cells as possible), we had some concerns about immune response to our EBV vector, we needed to pick a final output and we needed to decide whether loss of function in APC was the only marker we wanted to check for.
      In order to make sure our system could detect cancer with as few cells as possible, we realized that we would need some method of amplifying our signal to start producing output.  Luckily this task didn't prove too difficult as Molly was able to find several papers describing a modular signal amplifying system that had been used to produce large amounts of GFP but that we could likely modify to produce whatever we decide to be the output of our system.  With that, the open question remains as to what our output will be.  We think that something as simple as a combination of excreted and membrane-bound fluorescent proteins or luciferases could work but it will depend on where in the lining of the GI tract tumors tend to form (excretions from cells on the inner lining will be much easier to detect than those on the outside); we are still researching this topic.
      Another of our concerns centered around the EBV vector we plan to use.  We were concerned that, especially with much of the population having already been exposed to the virus, there could be a strong enough immune response to destroy our circuit before it ever reaches the target cells.  With a little more research from Katelyn however, we found out that because of modifications to the vector form of this virus, the natural immune response tends to be very low and should not threaten our delivery.  Having bypassed the immune system, our remaining delivery concern was transporting our vector safely through the digestive system.  We believe that we have found a capsule that will remain intact through the digestive system and dissolve afterwards that could house our vector but we are still researching that.
      The final major concern was whether we wanted to focus solely on loss of function in APC.  Natalie provided us with an article that discussed some other common markers of GI cancers that we looked through and considered.  Many of the markers discussed were, like beta-catenin, transcription factors with abnormal activity levels so we thought it wouldn't be too difficult to incorporate them into our existing circuit.  We also realized however that our original circuit had some room for improvement.  The design we presented for tech spec had the possibility of a false negative in the case of a cancerous cell that is constantly being exposed to Wnt - in that case there is no way to tell whether the CTNNB destruction complex is not forming because it shouldn't (Wnt is present) or because it can't (APC is broken).  With some more research we found out that over secretion of Wnt is another common phenotype in cancerous cells so we've decided to focus on improving our original design by accounting for this other possible error in the Wnt signaling pathway.  As of now we're still in the brainstorming phase but I'm pretty excited about some of the ideas we've thrown around.