Too Much Information (EW OMG TMI)

This week was truly centered around the discussion with the experts, and with good reason: the experts facilitate research and progression to an astonishing extent. We went from thinking about a species of cyanobacteria and devices within it to considering an entirely different genus, Vibrio, and its applications in the gut of a fish. That's a huge shift in the focus of our project, and considering how deep we are into the semester, it's a little concerning.
As resourceful as the experts were, (and they really were, we have tens of pages to prove it) I feel like it was too much information!! Well, I'm exaggerating a little, as there's never too much information when creating an engineering project based off of the research of other people, but I still feel overwhelmed. This fish gut idea is cool and Natalie likes it, but can we really pull it off?
We're going to have to identify a species that'll function the best in this unfamiliar genus territory, characterize everything it'll need to thrive in the ocean or the gut, identify devices and parts to augment it's decomposition functions, and have something comprehensive to present in two weeks. Can we do it? Then again, were we that far advanced in our research of cyanobacteria? Well for one, we knew there was a near-fluent parts transition between our former e. coli concept and cyanobacteria, but what now?
Our group has taken nothing off of the table as of yet, and we still have to consult the cyanobacteria expert on Tuesday.
I wish I had a way to gauge our progress against some kind of standard, so we could know if we were on track for presentations or a little lacking. That would be supremely comforting.

Experts! Ideas! Microplastics!

This week, we had the chance to talk to a couple of experts about our project. The first was an expert on the ocean ecosystem. She agreed that engineered cells might be able to mitigate the plastic pollution in the ocean, but she was quite concerned that the strategy would create a new problem. I suppose that's true, but the current pollution is literally killing everything in the ocean. Perhaps our project might disrupt the ecosystem, but at least there would be an ecosystem left to protect afterwards.

The second was an expert on using marine bacteria to degrade plastic. He was a) very enthusiastic and b) funny! Good to know scientists like him still exist! He told us about how the natural microbes degrading plastic might actually be bad for the environment, because they create so many microplastics. He also suggested some cool ways to make existing plastic more biodegradable, by adding trace nutrients like phosphorus, nitrogen and iron. However, I thought the coolest thing was all the work he has done studying the top micron of the ocean, and the plastic which acts as an extension of that. The bacteria up there have some really clever ways to adhere to and thrive on plastic!

So I think we need to make sure our project focuses disproportionally on microplastics, because we don't want to be generating more microplastics than we degrade. We also might need to move away from polystyrene, because it seems to degrade in UV light anyway.

The second expert also mentioned that microplastics seem to accumulate in fish and marine life. I had a crazy idea: what if we used the "E. Coli of the sea" to degrade plastics inside fish? The plastics would already be accumulated, and we would hopefully save a lot of fish from a death by plastic. Plus, the fish can do all the mechanical work of gathering plastic and expelling products of degradation, so we can just focus on the chemistry!

2 Phone calls and 99 Problems

When we were on the phone with the Wnt signaling expert, I was actually impressed at how our circuitry was withstanding his review. Initially, I was certain the whole thing was going to fall apart, but actually our circuitry did a pretty good job. Then suddenly wrenches came flying out of nowhere into our project. Apparently stem cells, naturally have high levels of Wnt as a natural part of their development. So while our circuitry detected cancerous cells, it also was going to produce a false positive for all stem cells. Several times now we’ve learned something in 7.013 and then applied it in lecture the next day. Professor Sive has yet to disappoint. Thanks for Professor Sive, we just learned all about stem cells in lecture. According to Professor He, the stem cells and cancerous cells are nearly indistinguishable, so we certainly have our work cut out for us. Somewhere we plan on starting tomorrow is that stem cells uniquely divide their mitochondria asymmetrically. In order to do this there must be some transcription factor or unique signal cascade being produced somewhere.  We plan on taking advantage of this and setting up a detection mechanism for this unique factor and finding out what exactly this factor is to help us see if a given cell is really cancerous or if it’s just a stem cell being mistaken as cancerous.

When we were talking to the virology professor, he didn’t seem to optimistic about using our EBV virus. He didn’t seem overall pessimistic about our project, which is at least a sign of hope. He gave us a couple of suggestions that will definitely help to guide us along in our hunt for the optimal vector.

I know I started out the course by saying I was truly hoping we would get to produce something unique and meaningful, and it seemed like it might not be possible to do something like that after our talk with Professor He. Our one shining ray of hope lies in whether or not we can sense the difference between stem cells and cancerous cells. If we’re able to do this and make our project an overall success, we might be able to produce a meaningful project.  While it’s extremely unlikely we will be able to make a legitimate contribution in the field of cancer detection, I would be extremely honored to know I helped inform doctors earlier of even a few cancer patients’ condition. To know that I had a meaningful impact on someone’s survival would be incredible. One day when I have more expertise I hope to be able to visit biological issues such as these and be able to increase someone’s chances of survival. 

Synthetic Biology and Bad MATLAB

Have you ever worked with a student who just can't seem to write good MATLAB code? Bad MATLAB tends to name variables with uninformative labels, and reuses variables for lots of purpose. It's often called "spaghetti code", because whenever you change one thing in the code, it affects things all over the program. Good MATLAB code only uses variables for a single purpose, so that changes can be made without far-reaching consequences. So what does this have to do with synthetic biology?

Let's think about molecular signals as variables. Nature isn't an engineer, so nature didn't really care to name molecules in any sort of logical manner. Neither 'LasR' nor its amino acid sequence reveals anything about its purpose. But we are engineers, so we ought to do better. If we want to really progress the field of synthetic biology, we should find some way to logically choose signal molecules that indicate their own purpose to the engineer.

More pressing however, is the extensive reuse of signals. Again, nature designs life in a highly iterative process, and doesn't care much to use unique signals. Instead, we should ensure that signals are only used for a single purpose. First, this requires building a family of molecules that doesn't affect nature, yet is completely absent in nature. Second, this requires rigorously adhering to a code of unique signal assignment, so that different groups don't use the same signal molecules.

Like in MATLAB, we can significantly ease the engineering process for biological engineers by separating concerns. Unlike MATLAB, we'll never have debugging tools quite as good as just dropping the semicolon...

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.

Success Seems Just Around the Corner

When we chose our project after the three ideas presentation, I knew we were in for a challenge when we decided we were going to try to come up with an early cancer detection method. I was explicitly concerned I wouldn’t be able come up with an creative solution to an issue that billions of dollars of research hadn’t already tried to come up with. While the Wnt signaling pathway is very well studied, perhaps we might have approached it from such an angle that no research group has thought to approach it the same way as us. Even we won’t actually build our system, I wonder if there would be a way to suggest this to a research group to test our system out for us? On the one hand they might appreciate the idea, but on the other they may not want to be doing a bunch of freshman’s projects.

I was overall really happy with the results from the Tech Spec Review. A lot of the feedback was very helpful, but it was almost all stuff we already knew to be thinking about. We were already trying to decide whether this should be an at-home procedure or just something you would do in a doctor’s office, and whether or not our viral vector would encounter an immune response from the body that would nullify our treatment. While the Three Ideas presentation essentially let us know we had a lot of work to do if we wanted to get anywhere with any of our projects, the Tech Spec let us know we were doing well, we just had to keep at it.

The largest issue we had been encountering was the issue of the false negative we were getting. When we realized we had left room for a false negative, it gave me a small feeling of panic. If we weren’t able to resolve our issue, we would have to back track immensely and re-do a lot of our work. However, I continuously underestimate our ability to come up with creative solutions to problems as a group. As individuals it would have been hard to solve this, but it took our group maybe 30 minutes tops of staring at the issue to come up with a viable solution. We were able to propose a clever microRNA destruction mechanism for a constitutively promoted output, where the output would only be expressed if Wnt enhanced the constitutive promoter. I was impressed by the ingenuity of our group, and I know we will be able to produce an incredible final project. I look forward to our final project and the other team’s final project.

Which Output Though?!

The wnt pathway is finally coming together (somewhat).  We are beginning to debug our original system in the hopes of finding a more efficient and reliable system.  Finding a way to reduce/eliminate false negatives, exploring proper marking, signaling, and output, as well as simply improving the pathway are our priorities.

I have been focused on the output stage of the wnt pathway, trying to figure out the most plausible output mechanism.  Originally, we went with generic GFP as it seemed to do everything what we wanted, and most importantly was easy to analyze.  The introduction of additional genomic sequences or markers then altered this original idea.

We chose to include topflash in our design, which uses luciferase as an output instead of GFP.  Luciferase has many benefits over GFP, including most importantly, the better ability to be amplified.  One of the problems we have is the fact that if only a few cells are detected, the fluorescent output may be hard to analyze.  Amplifying the output will solve this problem, and using an output that can be easily manipulated such as luciferase is to our benefit.

There is still a lot to do, but what we have accomplished so far is quite astonishing.  I can't wait to see what will be the eventual outcome of our project, and what the next steps will be.