Week 12: Wrap up and Reflections

This was the last official week of the senior project, and this will be the last weekly update for this blog. I will still be working on my project beyond the scope of the senior project at BASIS, but since this is an important milestone, this week I will write about my experience as a whole and what I learned from it.

This project and the internship that I have has been one of the coolest things I’ve ever done. I knew that I enjoyed scientific research before this, but this whole experience has really taught me what science is, why we do it, and how to do it.

I started off this project not really knowing what I wanted to do, and so I was really lucky to find a lab where I could help on and take ownership of a real research topic. I was excited to work on any project, but I’ve become quite attached to the crazy Frankenstein stuff that I’ve been able to do over the past 12 weeks.

In the past, I’ve gained some experience doing lab work and learned some useful lab skills, but this was the first time I have been fully immersed in a biological wet lab, and thus I did learn a huge amount about lab techniques and lab work. There is a lot of skill and precision required in doing experiments, and I definitely learned the importance of doing things the right way.

But more importantly than that, I’ve learned that even with all the precision and skill required to perform precise experiments, research is really driven by curiosity, creativity, and an open imagination. I’ve realized that science is not the straightforward method that we learned in school, where we have clear, falsifiable hypothesis that can be rigorously tested, rather it’s a pretty messy process where ideas come from anywhere and always end up somewhere completely different.

I’ve also learned the sheer breadth and depth of research topics, even just in a single department: while our lab is focused on using the best and most expensive imaging techniques to learn about life, the lab right next door is trying to engineer cheap tools for budding biologists in third world countries. I’ve worked in labs with fossils, eels, and worms, and each time I felt like I barely scratched the surface of what I was looking at. This is actually really encouraging, because I know I will always be able to find somethings to study and be curious about.

This project has at times been frustrating and slow-paced, and I’ve experienced lots of set-backs, as is expected in research, but I’ve really found something I love to do.

If you would like to see what comes out of my project, I will write a short research report that I will upload to the blog and will be presenting with the rest of the senior projects on May 23.

Finally, I’d like to thank my BASIS advisor, Dr. Sharma, my mentors at the Wang Lab, Chew and Professor Wang, Ms. Belcher for all the work she’s done for all of the senior projects, and everyone else who helped.

Thanks!

Christopher He

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Week 11: New Projects

Over the past few weeks, I’ve been mostly helping out on other projects in the lab while our chimera grow and we feed the new worms RNAi. The chimera are fusing quite well so far. One of the controls split after we released it, but the other 4 fusions are still alive.

Here they are on day 1 of the fusion:

The top 2 and the bottom left are sexual-asexual fusions and the bottom middle and right are asexual-asexual fusions. Junior 2 ended up splitting due to a poor fusion. It’s still early, but all of these fusions are displaying the same characteristics that we saw in the previous ones. They are forming clear midlines where the fusion is, that seem more pronounced on the chimera.

After 5 days, they look like this:

As you can see, they are all pretty well fused already and seem to be healing even faster than the previous batch. There is some residue and rejection that you can see in the petri dish, in line with what we saw before.

Meanwhile, we worked on two other projects happening in the lab. The first one was inspired by a paper by Arnold et al. that showed that a change in the microbiota of a planarian, i.e. the bacteria that live in or on the planarian, resulted in a change in tissue regeneration by activating a certain immune signaling molecule. We wanted to see how other bacteria might affect the regenerative abilities of planarians, so the idea is to culture different kinds of bacteria and inject those cells into the guts of planarians that have been cut and see how that affects regeneration. The culturing of the bacteria is relatively easy, the harder part is the injection. We use a tool called a microinjector that can precisely inject solutions into organisms or cells. Over the past few weeks we have been playing around with the microinjector, and figuring out how to inject into the planarian gut, which requires some dexterity and precision.

The other project we are working on involves altering the gene expression in planarian stem cells. We are essentially trying to change what genes are activated in planarian stem cells and see how that affect the organism. How we plan to do this is engineer stem cells with altered gene expression and replace the existing stem cells in a worm with the engineered ones. We are working on the replacing and imaging component, and the first thing we want to do is figure out how to label specific cells and see the gene activity in those cells. We are using a method called WISH, whole mount in situ hybridization, which is a pretty common technique for labeling gene expression in situ, on site, so you can see where a gene is being expressed on a cellular level. This is a very involved process that includes fixing the worms and hybridizing a probe to the gene of interest and takes 4 days to do. We’ve spent the last week putting together the materials and reagents that we need and creating the probes that we plan to use, and will do the actual WISH next week.

Thanks for reading,

Chris

Week 10: Fun Stuff

Over the past two weeks, most of the work has been in preparation for the next round of chimera. We have made several fusions that are around a week old at this point, however I won’t have pictures of them until next week. I do have one picture of the most amusing chimera, which curled around itself to form a doughnut shape.

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Our Chimera Doughnut

I’ve also been doing some other fun side projects over the past few weeks that don’t relate directly to the chimera.

One thing is Foldscope, a project my advisor helped on previously. Foldscope is a paper microscope, a product with the aim of providing everyone with an affordable microscope. Foldscope is a very intriguing product, that is both easy to make and relatively functional. Paper makes up essentially the entire structure of the microscope, and the only other piece is a small magnifying lens held in place with self aligning magnets.

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credit: foldscope.com

It’s very easy to put together and intuitive to use, and has the ability to attach to smartphone cameras and project images onto larger surfaces. The main engineering concern with microscopes, stability, is arguably the least robust part of the Foldscope, but given its price and ease of use, the usability is quite high. Here are some pictures I took using one I put together in less than 10 minutes:

 

 

Another interesting side project that we have just started is seeing how bacteria affect the regeneration of planarians. As I’ve talked about before, planarians have an incredible regenerative ability. This ability likely has many contributing factors, and a recent study posited that bacteria could play a role. What we want to do is try injecting injured or cut planarians with different types and concentrations of bacteria that we have available in the lab and seeing how that affects their ability to regenerate. This could also lead to some insights on how animal microbiomes affect their immune and regenerative systems.

We have just started to play around with injecting planarians with bacteria, which involves using very thin capillary tubes to puncture a anesthetized planarian and inject a bacteria solution into its gut. I will also have pictures of this set up next week.

 

Thanks for reading!

Chris

Week 7: Ralphy!

This week, we saw some really cool things happening!

Most of what I did in the lab this week was rather routine: on Wednesday and Friday we fed a new batch of planarians the PC2 double stranded RNA that we had created to see if we had good dsRNA and to prepare for the next batch of chimera. On Wednesday we also started to expand a population of asexual planarians that we had. This is done by cutting the worms in half and waiting for the halves to regenerate, effectively doubling the number of worms in that population over the span of a few weeks. Taking care of the worms is actually relatively easy: they need to be fed mouse liver once every few weeks and their water needs to be cleaned and switched, just like for fish. They do occasionally get infections, but they aren’t super harmful and can be dealt with with more careful maintenance. With proper care, the planarians can become quite big: there is one box with worms 10x the size the ones we are using.

Now for the interesting stuff:

We have three successful fusions, which have survived more than 2 weeks now! Two of them are chimera (sexual-asexual fusions) (aka ralphy) and one of them is a control (sexual sexual). Out of the two chimera, one was done with an uneven cut, i.e. the asexual portion was much smaller than the sexual portion. This may be significant, as we saw phenotypic variations between the two.

Here is what they looked like right 4 days after fusion:

 

On the left is the sexual-sexual fusion. You can kind of make out one eye on the right side, but for the most part, there are no discernible body parts. However, the fusion is pretty good already, there is only a faint mid-line separating the two halves. On the right is the ralphy, sexual on the left. As you can see, the fusion is much less clean than on the the control. There is a clear separation between the two halves, its pretty curled up, and their heads are still somewhat separated.

Soon after, we took these images:

 

On the left is the chimera, 10 days after fusion. As you can see, it has spread out much more and actually has grown an extra eye. the two halves aren’t as uneven as before, even though there is still a clear mid-line separating the two halves. Overall, looking great! On the right is the control, 6 days after fusion. Already, you can see how it has formed more clear body parts and has grown an eye! The mid-line is already starting to fade a little, even though it had less time to heal. This is really interesting, and could indicate that the tolerance for fusion could be higher between the same strain.

How they look now, after 15-20 days!

 

Here we can see a marked difference in all three! In fact, they have even started swimming, which is pretty crazy. The control, on the left, has very little mid-line left, and even though it still only has one eye, it seems very healthy. The small Ralphy in the middle shows some really interesting features: There is almost no discernible mid-line, even less than the control, but you can still make out the color difference. This could be an indication that there is a threshold for immune responses, and because the asexual half was so small, the response was much smaller. On the right, we see our 3 eyed ralphy, which has a pretty significant mid-line, but is swimming around happily as well.

 

Another thing we saw was rejection of some cells from the chimera, as you can see above. These are groups of cells that the chimera ejected, however we do not yet know why or what causes this to happen.

 

These initial results are pretty fascinating, and hopefully we can see more of this as we ramp up our experiments.

 

Week 5&6: Successes and Updates

Hi everyone!

As promised, today will be a full blog post about what I have done over the past 2 weeks, as both of these weeks were filled up with non-senior project related activities (robotics) and I was only able to complete one week of work.

The past two weeks have been mostly more of the same.

My mentor was busy with a project in another lab last week, Continue reading

Week 5: Quick Update

Over the past week and during the next week, I will be spending most of my time at robotics competitions with my team, the Popcorn Penguins. Unfortunately, that means I will not have much time to work on my project over these two weeks, most likely I will get a combined one week of work done. Because of this, I will write a double-blog post next week detailing the progress of my project over the these two weeks.

I plan on creating more chimera this week to perfect the protocol and also try to observe the phenotype and behavior of the control and chimera that were successful last week.

Meanwhile, here is a picture of our robot!

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Week 4: Bio is Weird

Biology is weird. Things often don’t work when you expect them to, and they do work when you expect them not to.

This week, we tried a second round of essentially everything we did before – chimera, dye testing, and double stranded RNA synthesis. All three seem to be working much better than before!

We dyed the worms on Monday with a new dye called CFSE, a flourescent dye that is often used in C. elegans to track lineages. As it is able to persist on a generational time scale in C. elegans, we thought this dye would be able to persist in our planarians for the few weeks that we needed to study the chimera formation. We dyed the worms for a few hours and fixed them on filter paper as we would a chimera. On Friday, we imaged the worms and found that the dye had worked very well, as yo can see below (the bright spots are signals from the dye).

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Dyed Planarian

Later on in the week, we tried a second round of chimera. This time we only tried 3, two chimera and one sexual-sexual control. This time, after we fixed them, we took extra care not to leave them for too long to dry out. We found that after just 1-2 days, 2/3 were not only still living, but seemed to have fused! Hopefully they will heal well enough so that we can release them soon!

That’s about it for this week, next week hopefully we can see some more results and start perfecting our methods!

 

Week 3: Getting Ready for Round Two

Our first round of chimera were unfortunately unsuccessful, almost certainly due to my inexperience (*incompetence*). For this week, we prepared for the second round of Chimera by treating 20 more sexual and asexual worms with the PC2 RNAi knockdown.

We also tried making more PC2 on our own, which involves amplifying a template DNA through PCR, transcribing that DNA into RNA, and annealing the RNA strands together to create double stranded RNA. We also managed to slightly screw this up, forgetting a questionably important dilution step, resulting in a yield four times what was expected, but a solution that may be completely useless. We will still use this likely defunct solution in a future RNAi, because science (who knows maybe it will work better).

 

In this blog post, I will go into full detail into the chimera Continue reading

Week 2: The First Chimera

My project has begun in earnest! After a much less busy break week where we began some experiments with dyeing the planarians, and preparing some more worms for chimera making with the PC2 knockdown, we have finally started to make our chimeras!

We made our first chimera on Tuesday with the worms that we had knocked down the first week. Making the chimera was relatively straightforward – we placed the worms on a cold surface to keep them from moving too much, and put them under a microscope to see it better. Since this was the first batch of worms, we started with a few control experiments, where we cut a single worm in half and pushed the two halves back together to fuse. After the two halves are pushed together, we lock them in by placing a wet cigarette rolling paper on top and box them in with filter paper on each side. This keeps the halves from running away from each other, which they tend to do, for some strange reason. In the past, only worms cut longitudinally (cut from head down to tail) were able to from chimera, but we wanted to see if laterally

cut (cut off head from the tail) could work as well, so we tried a several of those as well. After boxing them in, we placed them in a 10ºC incubator.

We also tried making some more chimera on Thursday that combined different ratios

Continue reading

Week 1: Playing with Planarians

My internship has finally started, and the first week has been really fun.

I started the week off with lab safety training, where I went through the necessary procedures for maintaining safety in the laboratory setting. After a meeting with the building’s safety coordinator, I was all set to start my project.

The first thing we did was in prepare the planarians for making chimera in the coming weeks. To make the chimera, the planarians need to be immobilized. We do this via a method called RNA interference (RNAi). This is a commonly used method where double stranded RNA that is used to block a certain gene from being expressed. The gene we want to knockdown is PC2, which is a neuron protein that is important for motor neuron activation in planarians. We use the RNAi to block this gene essentially as a way to temporarily paralyze the planarians so we can perform our experiments. Doing the RNAi was fairly straightforward: we mixed the RNA solution with some crushed up liver and fed a group of both sexual and asexual Schmidtea. It will take more than a week for the PC2 knockdown to take full effect, which is why we have started it so early.

After we had finished setting that up, I was finally able to spend some time playing

Continue reading