Team 99 Bottles Reaches Shore

So, the journey has finally come to an end. Throughout this semester, this project definitely has had its ups and downs. There were times when I got really excited and times when I got really frustrated, times when I questioned what the goal of the project was and times when I thought we could maybe save the world. But I imagine that’s all part of the journey. I suppose roadblocks and incompatibilities are occupational hazards of bioengineering, and fun and discoveries are the rewards. Maybe it’s because I’m new to the field, or maybe it’s because the field itself is relatively new, but it seems to me like the rules still have yet to be defined. It seems as if you can do whatever you want as long as you have access to resources and stay away from anything ethically controversial. The possibilities are endless, and that’s exciting but also challenging because that means there are fewer examples to go by, fewer guides to help you know what you are doing.

Indeed, I thought that the freedom of the project was one of the hardest things about this class, but also one of the most valuable elements of the class. It’s made me realize that although high school has taught me how to study for exams and maybe even how to learn, it hasn’t taught me how to do research. I think that’s why this class has been one of the most novel experiences since coming to MIT. It asks you to learn without a defined curriculum.

Furthermore, I loved getting to know the people in my team. Each and every one of them was amazing and brought so much energy, curiosity, ideas, and hard work to the table. We went through a ridiculous amount of changes together, from salt water to fresh water and E. coli to Vibrio. As I continue on in course 20, I really hope that I will gain skills that will make this type of work easier, so that one day I can maybe do something of this sort for real (if the rigor of research doesn’t scare me away). Overall, it was a great introduction to something I had never been exposed to before.

All the Feels

It's been a fun, informative ride. We got to experience synthetic biology techniques first-hand, transforming e. coli into banana scents and light sensitive printers. We examined the social problems our field can face, with relation to bioethics and public opinion on our topic. We played out the economical difficulties that can prevent the acquisition of other researcher's parts, which is apparently a huge barrier to bringing products to market. Most thrilling of all, we we're all placed in the shoes of a researcher and told to use our brains and synthetic biology to affect great change in the world. No other course at MIT offers the 20.20 experience, so I'm really glad I took the plunge.

The process of collaboration with Team 99 Bottles was pretty amazing, especially as members got comfortable and let their talents shine through. Colin emerged as the lead researcher, Lee was the organizer and editor, Jackie was secretary who'd catch our oversights, and I just kept my head in the clouds, blurting out novel ideas from time to time. I'm not sure if there's an ideal group of people to comprise a research team, but Team 99 Bottles was certainly on the right track.

I feel Course 20 knowledge flowing through my veins! The concepts and methods by which we manipulate bacteria seem much more familiar and accessible than they did before. Device diagrams and timing diagrams have actual meaning! And the process of researching and uncovering information? Not as terrible as a thought it'd be hahaha. On that note, experts are certainly an invaluable resource, reducing our research times by a tenth, at least. That must be a great feeling--to have mastery of a subject to a point where you can be consulted for your knowledge and help others accordingly. 

It's funny that I ended up declaring Course 2 when I was inclined to Course 20 at the beginning of the year. It has nothing to do with me being scared away our put off by 20.020, but more about the variety of options post-undergrad. Biotech is an emerging field and I'm not sure if I want to work in academia yet, and besides those two options, the Course 20 road seems a little limited. Course 2 on the other hand have a multitude of career options to choose from. I suppose I'll know if my decision was right in due time, but I'm so appreciative to the 20.20 experience for informing my path much more than I could have done alone. Natalie has been a wonderful teacher and mentor throughout the entire experience, guiding us with a gentle hand and a great smile. Between the senior mentors, my peers, and Natalie, I feel like I have access to a wealth of resources and informed people as I move forward in my college career, and I'm so grateful for that. 

To conclude, I no longer think bacteria are gross (as I alluded to in my first blog post :) ). There's great potential in synthetic biology to solve world problems and great potential in each and every one of us to see it through. Thank you everyone for your great collaboration to the very end! Thank you seniors for being so charitable with your time. Thank you Natalie for taking us on this ride and I wish you so much luck with your future endeavors. :,)

Final Thoughts

20.020 is over! I am happy and sad, and proud of what my group accomplished. It is so strange to look back at past blogs, getting a peek into my brain at that point in time. I almost don’t even recognize my own thoughts. Looking over the old blogs, I decided that 20.020 was one of the most worthwhile classes I’ve ever taken. It reminds me a lot of Terrascope, but taught in a smaller and more focused setting, which I preferred. Both projects I undertook this year were incredibly complex, much more so than I realized at first. In both cases, there was no way to account for all the possible variables; we just had to pick solutions that seemed like they might work and improve on them as we went. Improving—that was an enormous part of design. More so than for the other group, our project went through a number of iterations as we went. First it was some sort of plastic decomposer that we might use in the landfill or oceans or who knows where… It later morphed into an E. coli with a salt balance kill switch, then a cyanobacteria equipped with plastic degradation enzymes, and finally a vibrio bacteria that we used to decompose additives instead of plastics themselves. I could never have predicted our final product at the very beginning of the process, and from the end looking back at the beginning, our idea seems so simplistic and doomed for failure. I could probably come back next year and improve on our idea even further—I wonder how long it would take to crystallize into a project that a “real engineer” would actually work on. I put “real engineer” in quotes, because I don’t think of myself as an engineer. But really, I just need more practice. I never thought we would be able to come up with the project we did; it makes me wonder what else I can do that I think I can’t.

The Struggle was so Real, but so Worth It.

Seriously though, we have come so far since the beginning of this class!  Looking over the posts from the beginning of the semester really provided me with some perspective of how much more I now know.  Having very little experience in such a huge field coming into 20.020, I had no idea what to expect.  Although we only touched the surface of such a large science, I feel as though I now have a better understanding what biological engineering is all about.

This class, for me, was not entirely geared towards the introduction of biological engineering however.  It also allowed me to work in a small group for many weeks, constantly re-engineering, fine tuning, scrapping ideas, and thinking of new ones as we strove to come closer to our ultimate goal of making early detection of cancer more accessible.  The skills I have learned in this class including proper research techniques, efficient time management, and complex thinking in general will certainly benefit myself while at MIT.

Once again, looking back at my previous posts, I can see a trend of a much more focused approach toward the end of the class.  In other words, the first few weeks of the class were simply exploration, finding things that fascinate us.  As the course progressed however, I found myself becoming more and more immersed in the ever evolving problems at hand, of which there seemed to be a new one each week.

How far we have brought our project, from three adventurous ideas, to one finely tuned proposal in such little time brought tears to my eyes (not really, but it was still pretty cool).  Not only that, but having our final project be as detailed as it was, without even having access to laboratory equipment really highlights the power of in depth thought and problem solving.  This class was awesome (especially you Natalie), and although it was a race to finish everything at the end, the final outcome was well worth the struggle.

The Semester in Reveiw

It's strange to think that the semester has finally come to a close. It feels like it went by so fast! And yet, I reflect on the amount of work that went in to our final project and realize that we actually spent a long time developing this system. I kind of can't believe that we finally managed to understand enough to put together our project, and yet, here we are with a "finished" presentation (I'll get to why "finished" is in quotes in a second).

I looked back over my very first blog post a few days ago, and the general gist of it was along the lines of "haha what's a cell?" I knew absolutely nothing coming into this course. Biology itself was a foreign concept to me, not to mention the idea of applying biological concepts to engineering. I had no idea what I was doing. I am so happy to say that that has changed. I feel confident now that I understand the foundational ideas behind bioengineering, and I am excited to continue learning as my career at MIT proceeds.

I'm really amazed at how far our project came over the past few months. The night before our presentation, we all virtually met up and went over our documentation and systems until we felt solid about the design. Even though it was late and we were all tired, the enthusiasm about our system never deflated, and it was so exciting to see the minds behind the project continue to chug along until we were confident in it. It was then that everything we had learned over the semester was applied as we checked through the details of our work. Seeing it all culminate was an incredible experience and it really brought everything in the course together quite nicely.

Now, you will notice that I put "finished" in quotations in the first paragraph. If I can sum up my learning during this course into one lesson, it is to never assume that I know everything. It seemed like every time I got attached to an idea or concept, someone else had a novel approach that was even better. With the viral vectors, for instance, my attachment to EBV was shattered when Dr. Coen suggested seeking an alternative, leading me to find oHSV, an even better match for our project. This inability to know everything is definitely not a bad thing, don't get me wrong. It means that there is always more to discover. The field is ever-changing, and there is so much more we could do with this project. Sadly, the class has come to a close, but that knowledge and thirst for new ideas will continue on with everything I do at MIT. Thanks for an amazing course. It's been a blast. Team Turtle for life <3

... and this is why we have experts ...

      When I wrote my last post, I mentioned that we were adding an extra component to our system to detect another possible mutation in the Wnt signaling pathway.  After thinking it over for a bit, we thought that we'd come up with a pretty nice solution and it seemed like our system was well on its way to completion.  On Wednesday, we were scheduled to speak with some experts on some of the things we're working with - viral vectors and Wnt signaling - largely hoping to confirm our ideas and maybe get some thoughts on ways to improve moving forward.  We got quite the wake-up call; there's a reason we talk to experts.
      It turns out that the vector we were planning to use was not quite as well characterized as we had thought.  It definitely had the capacity we thought it would but Dr. Cohen (our virus expert) had not heard of it being used effectively without pathogenic effects.  Furthermore, this particular vector is not suited to be freeze dried and delivered by capsule as we had planned due to the weak lipid membrane that surrounds it.  Luckily Dr. Cohen was able to suggest some other possible vectors that Katelyn is doing some more research on.
      Still reeling from our first consultation, we prepared to speak with Dr. Xi He - an expert on the Wnt signaling pathway.  I tried to stay confident that we had done enough research for this second consultation to go well but then again, we had been really confident about our information going into the first.  Fortunately Dr. He was able to confirm much of our research into and reasoning about the pathway, and broadened our list to the 4 most common mutations in the pathway that lead to cancer (all of which seem to effect beta catenin activity).  We did run into some problems however when we proposed our entire circuit - that is, a system which only produces output in response to sustained high levels of beta catenin activity.  It turns out that because intestinal cells have such a short lifetime (all cells are replaced every 5 days), beta catenin is almost constitutively active in GI stem cells, meaning they would be tagged alongside any hidden cancerous cells.
      The problem we then faced (and will still be working on for a while) is how to tell a cancer cell from a stem cell.  We believe that one of the points Dr. He made may have given us the answer.  Although this pathway is almost constitutively active in both types of cells, cancer cells lack the ability to control when it is active.  Each of the four genes he mentioned, when functioning normally, can either increase the activity of beta catenin or reduce it (at least to a first approximation).  So our strategy at the moment is to try to use our position of acting within the cell to test whether those genes are working.  We are trying to think of each gene as a stop button, each of which is wired up to beta catenin in a different way.  We will wait for beta catenin to be active for a "long" period of time then press each of our metaphorical stop buttons and check whether the downstream circuitry is still functional by looking for changes in beta catenin activity.  Most of our research over the next week or so will likely center around figuring out what these "stop buttons" might look like (if they can actually be made at all), but overall I am very excited to try to get this system to work.

Project 2.0

So it seems as if the calls with the experts have prompted both groups to readjust their project goals pretty dramatically. If I learned one thing, it's that I prefer talking to people over researching online. The experts were just so informative and helpful in such a short amount of time. I kind of wish we had gotten to talk to experts earlier, so that we could have picked a more appropriate chassis from the start, but I suppose there's value in exploring the waters on your own first and learning from your mistakes.

Looking back, I feel like we were trying to piece together a system that was logically sound but not necessarily the most intuitive. A lot of choices seemed arbitrary and involved compounds that we happened to stumble upon and seemed to work. After running through the design process once and talking to the experts, I'm starting to realize the value of drawing from the experience of others to obtain a better sense of what tools and parts are available and appropriate in the bio engineering world. 

I'm treating this next phase as our project version 2.0. Vibrio seems like a very viable chassis, and I'm ready to see what the cyanobacteria expert suggests. 

On another note, something that really excited me was the fact that the bacterial expert had been contacted by industry to create something very similar to our project. That means that our idea is viable and in demand! This project has me seriously considering pursuing something similar in a lab as a UROP - I think that if I could work on a project like this in a team and have some control in the direction of the project, I would be working on my dream UROP. 

So Much Can Change in 30 Minutes

On Wednesday, we talked to experts on the topics pertaining to our topics. We thought we were pretty well prepared going into the meetings. We had several questions written out for each of the experts and knew how to present our project to them.  We even knew how to pronounce their names (this was a larger problem than we had anticipated though).  We had people ready to take notes and were open to any information we could obtain from these people who know much more about Wnt signaling and viral vectors than we do.  There was one thing we were not completely prepared for:
"Yeah that is not going to work."
Neither of the experts actually said those words.  They were much kinder and more scientific, but that was the takeaway.  We learned from he viral vector expert that our choice of a virus was not ideal.  When we asked him for suggestions for a replacement, we were again reminded on how much we do not know.  We need to know how long our sequence is and what cells and how many cells we need to infect before we can really decide on a vector.  From the Wnt signaling expert, we found out that one of our issues will be that many of the cells that cause GI cancer are stem cells and that intestinal cells have a life span of about 5 days. There are also four main causes of GI cancer and we had not considered two of them.  After the conversations with the experts, we discussed a new system and each have areas to research, so we have not lost hope.  However, I have definitely been reminded of how little I know and how much I just did not consider. Sometimes the enormity of the subject is frustrating because I know that I will probably never fully understand all of it, but I think that is why the impact of a project is so important.  Even though I know that we will never complete this project in a lab, I am still motivated to push forward with my ignorance and try to just learn whatever I can to create a usable system because I want to help people and for there to be a simpler early detection system for cancer.  Science is great and all, but the "why" matters.

Meeting with the Experts

This week was one of my favorite weeks in 20.20 so far! The experts we talked to were so helpful in answering our questions, probably saving us hours and hours of research. Up until this week, I had the vague notion that our system in its current form would not work, but couldn’t really come up with a definite reason other than that it was kind of a mess. (Natalie described it as a dam with a bunch of leaks that we kept trying to plug with our fingers, which I thought was pretty accurate.) While our system now has a less well-defined goal than before the meetings, I believe we are moving in a better direction now. Such is the iterative design process! It is a little frustrating to be possibly taking a large step backwards at this point in the process, but if it will help our design, that’s what we’ll do.

One of the most important points brought up by the experts was the harmful effects of microplastics and plastic additives. Although we knew we would have to worry about the by-products of decomposition, this concern wasn’t as high on the priority list as figuring out how to make our system work in the first place. However, I had no idea of scale of the real problem: the spread of microplastics to nearly every corner of the globe, their tendency to accumulate in the digestive tracts of marine life, and the detrimental effects of plastic additives such as BPA (which can mimic estrogen and disrupt the endocrine system). We may narrow down to a more specific focus, which we knew from the beginning was a possibility—our goal was so ambitious! One interesting idea we are discussing is to engineer some bacteria to live in fish and chow down on the microplastics they eat. It sounds pretty cool and I think we should run with it, but who knows whether it will work. I’m excited to talk to Danny Ducat on Tuesday, and to see what direction our project heads next!

Experts do know best...

This week was both the best and worst in the class.  We received much more information regarding our project--very helpful information that we never thought to research before talking with the two experts.  However, this information maybe wasn't what we wanted to hear (unhappy face).

Dr. Donald Coen first talked to us regarding our choice of viral vector.  He provided useful insight as to why EBV was good to use in theory, but it would be very difficult to introduce into the gut for a variety of issues.  He told us of the possibilities of other vectors (pox viruses for example) that may be worth our time to research.  After talking with Dr. Coen, we all felt as though we were back at square one--at least we had a wealth of new information to combine with our preexisting knowledge to create a perfect design.

Next, Dr. Xi He, an expert in Wnt signalling told us of the four most common mutations in the Wnt pathway that could lead to a cell turning cancerous.  This will be our other focus moving forward: finding out more about the big four mutations, and trying to implement them into our system.

The next few days will be full of us attempting to make last minute adjustments to our system with the suggestions proposed by the two experts.  I am confident we can get everything researched and implemented before the final presentation, and make the best system possible with the information we have thus far.

An Expert's Opinion and a Student's Shock

This week we had the opportunity to speak to two professionals doing work related to our GI Cancer study: Dr. Xi He, an expert on Wnt signaling, and Dr. Donald Coen, a virology department faculty member at Harvard. Both had incredible insight for our project and were able to provide a lot of great feedback for our team.

That said, speaking with them also made me wonder what on Earth we were doing.

Well, that is a bit of an exaggeration, but the point is that I had no idea how much we still had to cover for our project to even be close to a feasible solution. It's one thing to go online and read literature about a biological system, it's another thing entirely to hear from someone who has spent their entire life researching that system. Sure, both posed many questions we were prepared to answer, but many times their feedback led us to more and more that we still had to look into. For example, in my last post, I raved about the EBV virus. From Dr. Coen's input, it turns out that there are many far better options that I have to look into! Similarly, Dr. He outlined four clear cellular malfunctions that can lead to the development of GI cancer, which we had previously not looked into.

By no means is this a negative thing. Seeing how much work is still to be done was certainly shocking, but now we have the opportunity to improve our system significantly thanks to the opinions of the experts. Getting clear feedback was hugely important to making sure we were on the right track, and though we still have a lot to do, I think we are definitely getting there.

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.

That False Negative Problem

So whenever you test someone for some disease, you can have a negative, positive, false negative, or false positive.  As far as the falses go, a false positive is probably not that bad.  In the case of our system, that would mean that an output was produced when the person does not actually have cancer. After the output was produced, another test would be performed to actually find the cancer with a scope and it would be discovered that there is no cancer.  Although it would cause the patient a lot of unnecessary grief over their diagnosis, a false positive eventually just ends with the patient being relieved.  A false negative, however, is much more frightening.
Recently, our group discovered a really probable false negative that our system could give when interacting with GI cancer.  Since our system only produced output when Beta-catenin was present without Wnt, the cell would be free to have free Beta-catenin and Wnt and not show that it is cancerous.  If the presence of Wnt and free Beta-catenin was just an occasional occurrence in cancer cells and they would produce the output at all other times, this would not be a problem. However, some cancer cells suffer from hyperactive Wnt (too much Wnt).  Therefore, there would be no output would be produced, but the cell would be cancerous.
We are in the amidst of figuring out how to deal with hyperactive Wnt.  Our current solution is to have two systems.  One system is the old system and the other involves miRNA degrading a second output.  We have also looked at possible ways to use Wnt presence as a repressor and promoter for the same output.  This one-system system would create fewer parts for the cell to produce in order for the system to work, but may be less accurate.  This is a dilemma into which we are still looking.
This issue has made me think of the part of the project on which I was just working. I had to find a way to amplify the output.  I think that we are going to use the modular amplifier that I found even though we have to produce a few more parts.  This reminds me of the two-system vs. one-system debate because it forces the engineer to decide between a larger system built with modular parts vs. a simpler system that is tailored specifically to the issue/goal at hand.  Maybe the second way is better, but I am very grateful that papers have been published and the Registry of Biological Parts has been created to help make the whole field more modular and a little easier to grasp by people just learning things like me.

New Chassis

After receiving feedback from the tech spec, the team now has new direction: to find a new and improved chassis that won't die of malnutrition in the ocean waters.

We identified several candidates, but after much deliberation it was decided that cyanobacteria would, after all, probably make a better chassis than Lee would. So we've been looking into several strains that look promising. Exactly which strain will fit our needs best remains to be determined, and further researching that is our next step. After that is decided, it seems like we're just going to transfer the old device-level system onto the new chassis by looking for new parts that are compatible with the new strain of cyanobacteria. I find it pretty amazing that you can just transfer this kind of system, and here again the versatility of bioengineering, at least in theory, strikes me.

I kind of wish we could actually try this in a lab. It would be so cool.