The Final Countdown

Hi everyone!

I never thought I would say this, but thank goodness one of my experiments didn’t work out! The experiment in question was the one on RNAi, during which I would have to move (and count) 450 worms a day for three days. I put the rrf/tm homozygous line that I bred on the special RNAi plates, waiting for the 2nd generation whose genes should definitely be turned off (normally the 1st generation is not on the plates for enough of their lifespan to completely do so) to be in the 4th stage of life, but the 1st generation looked really, really sick and didn’t even lay eggs. Even though it’s not what we planned, it’s still an interesting observation. Often in science,  a lack of results actually turns out to be results  of their own. Because this experiment didn’t pan out, my prediction of having to spend so much time in the lab was thankfully wrong.

In other news, we performed qPCR to measure the gene expression of worms infected with 4 different pathogens (the 2nd repetition of this experiment). Unlike my first qPCR, this one’s results seem much more credible. The graph below shows the relative expression of the gene in question, nspc-14, in different lines and on different bacteria.

nspc14

HB (HB101) is the wildtype bacteria that are used to grow worms under normal conditions-our control. PAO1 is P. aeruginosa, a strain of this pathogen that is harmful just to worms. SM is S. marcescens and SA is S. aureus, both bacteria that are very pathogenic not just to worms, but to humans as well. N2 and TM are our main worm lines- N2 is the untouched wildtype, and TM3504 is a line that lacks the pqn-44 gene. nspc-14, the gene that is specifically being looked at in this graph, is hypothesized to work with and be related to to the expression pqn-44, both of which are thought to influence innate immunity. Given this, it makes sense that genes responsible for an immune response are upregulated on pathogenic bacteria (PAO1, SM, SA). It also makes sense that TM always has a lower expression than N2, since it is missing the pqn-44 gene. HB N2 is not included on this graph (it should be on the very left) because something went awry with the control for baseline gene expression, so it showed expression for every other gene to be very high, making the other bars look insignificant on the scale. I am performing the infection a third time, though, so we’ll have that data next week, hopefully before I fly out on Wednesday.

I’m so sad this is my last week!!! I can’t wait to come back to my family, but I’ll miss living in Warsaw and working in this lab 😦

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Busy bee

Hi everyone!

This last week in the lab has been absolutely crazy, and it’s not looking like it’s going to calm down next week. I’ve been spending 8-9 hours every day in the lab, and I still have so much work to do! In fact, I’m actually going to the lab today, too, even though it’s a Saturday.

So, you know how in my first blog posts I talked about how difficult it was just to transfer worms from plate to plate? I’m laughing at my past self for that, because last Friday, I started doing microinjections on my worms. Now, that, is difficult. Not only do I have to move my worm of choice to an agarose pad with a drop of mineral oil and make sure it doesn’t move, I then have to use a needle that we pull ourselves from a glass microfilament, so thin that you can’t even see the end without a microscope, and inject my victim with it in a very particular bundle of mitotic cells near the worm’s gonad. The first couple of worms that I tried to inject, not surprisingly, I murdered violently by tearing them in half with the needle or other methods of “how not to inject worms.” I think I finally got the hang of it this week, though, and I injected 9 worms in 3 hours, much better than the 3 worms I injected in 4 hours last Thursday or Friday.

It’s insane to me that I only have a week and a half left here, and it’s insane in the lab too. This week, I have to run 3 simultaneous experiments, not to mention isolating RNA from all of them later on and analyzing them by qPCR. One of these projects is the second part of the RNAi experiment, which involves me moving 450 worms by hand every day for 3 or 4 days, which is going to be fun. I am also going to keep doing injections, and do a 3rd round of infections. I did the 2nd infections this week, and I’m also going to have to isolate RNA from that. I wouldn’t be surprised if I spent 9-10 hours a day in the lab this week just to have the slightest possibility of finishing all that I have to finish before I leave. I’m already one of the first ones in the lab in the morning and one of the very last in the evening, so I’m totally looking forward to being even earlier and staying even later.

I hope you all are doing well!

Maybe my C. elegans were moody that day

“If you don’t get results, repeat the experiment two more times so that your failure is statistically significant.”

Like I mentioned in previous posts, some things don’t always work out as perfectly as you’d like, especially in science! Someone showed this at the lab meeting this morning and everyone laughed because they all know it to be true:

lab anxiety

So, anyway, we got my qPCR results back! And I have to repeat the experiment twice. We got results, but not the ones we were expecting. One of the samples had a much higher output than the rest of them when it had no right to be, and some had a much lower response when that protein should have experienced up-regulation. I might have messed up along the way, or these results could be accurate- we’re repeating it to make sure it is not the former. Since Poland, as a Catholic country, basically shuts down for 3-4 days around Catholic holidays, we’re going to start the experiments after Easter (the Polish word for Easter is “wielkanoc” which literally means “the great night.”)

I am also finally putting bacteria on my RNAi plates tomorrow, which means we can get started on getting results from that also when we come back on Tuesday. The next three weeks are going to be busy, repeating all the infections on 4 different bacteria and starting (and hopefully finishing) the RNAi one.

I hope you guys are having a great week!

Slow week in the lab

Hi everyone!

This week, I’ve spent very limited time in the lab (4-5 hours a day) because my grandma had to go to the hospital and I’ve been spending a lot of time visiting her, so I haven’t done all that much.

I’ve finished isolating RNA from both rounds of infections and DNAsed it so I have a purer sample of RNA, on which we are going to do qPCR (or real-time PCR) today. I’ve also transformed bacteria with three plasmids that we need for the RNAi experiment. Today, I’m going to synthesize cDNA, and tomorrow we might start injecting worms with the plasmid constructs that I made a few weeks ago.

Hopefully next week’s post will be more exciting!

I hope everyone’s enjoying their spring break- spring seems like a (very) distant horizon here.

What week is it?

I’m constantly amazed by how fast time is moving, it seems like just last week was my first day in the lab!

In the last week, I’ve set another infection of my wormies with two bacteria, B. subtilis, which is hypothesized to be completely harmless for C. elegans, and P. aeruginosa, the strain of which we are using is harmless for humans but very harmful for the worms. I’m very very glad that these bacteria don’t stink as much as S. marcescens (yet, at least), but P. aeruginosa has a very funky green color that makes it look kind of evil.

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I’ve also successfully isolated the RNA from the worms of my last round of infections with S. marcescens, which is harder than it sounds because RNAses (enzymes that break down RNA) are everywhere and they can’t be killed by bleach or ethanol.

The gel that I ran last week to determine if there was a line of double homozygotes from my cross showed that I indeed had as many as two lines with the right genotype! We’re going to use these worms next week in an experiment using RNAi (RNA interference) to disrupt the normal function of specific genes. Lines 5 and 15 are homozygous for both traits (there’s only one bar on the left side of the gel for those worms, the same as the TM line, which is known to be homozygous recessive for the pqn-44 gene, while 16 is heterozygous because it has bars from both the TM and rrf lines. On the right side we can see that all 3 lines are homozygous for rrf because they have the same bars as the rrf worms).

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Now that I’ve finally remembered to add pictures to my blog post (18 years later) I can show y’all what worms look like! This picture is what a normal plate looks like when it has a lot of worms on it– they’re tiny but you can see the little white lines with the naked eye, those are my C. elegans.

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When you synchronize worms so they’re all in the same stage of life, they eat the bacteria on the plate while moving further and further as a group, which as my mentor, Vlada, calls it, looks like a battle front moving further into enemy territory.

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This plate is a good example of what it looks like when it gets contaminated by other bacteria. It looks yucky and smells even worse, trust me.

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Next week, we’re doing the RNAi experiment and isolating RNA from the infection I set yesterday, so stay tuned to hear more about Martha’s lab adventures…

Pathogenic bacteria!

Last week, I worked with the first species of pathogenic bacteria that we’re going to be infecting my wormies with, S. Marcescens. Let me tell you something you might not know about pathogenic bacteria: they stink. They stink so bad I wanted to get a clothes pin or a binder clip or something to make my poor nose stop suffering, but I had to look professional and like it was no big deal because I didn’t want my mentor thinking I was silly. To add to this, apparently these ones only stink “a little” in comparison to some of the other ones that I’m going to be using. I’m really looking forward to next week.

When I was working with them, even though I was very careful and obviously had gloves and a lab coat on and was working very close to the bunsen burner to make sure everything remained sterile, some of the only thoughts going through my head were “I’M SICK ALREADY. I CAN FEEL IT, I’M INFECTED.” (Update: I am thankfully not infected with S. Marcescens).

Another project I’ve been working on is crossing worm strains to get a double recessive homozygote that has both genes of interest effectively disabled. It looks like we should have at least 3 lines that have that genotype–I’m running the gel that will tell me for sure whether or not they are indeed double homozygotes right now.

A couple of days ago, I asked my mentor when I should synchronize my wormies so I can infect them with the pathogenic bacteria, and she said “you decide.” She’s now given almost complete control of my project to me, myself, and I, which is kind of a terrifying thought. She’ll be there to help me and answer whatever questions I have, of course, but it’s becoming a more me-driven project vs. her teaching me all the methods up to this point. Last week, she let me choose the restriction enzymes by myself in order to make sure we have bacterial colonies with exactly the right inserts, and that went perfectly well, so we’ll see how this goes!!

I don’t think I’ve mentioned this on my blog yet, but another thing which makes my lab the coolest lab on the face of the Earth for me right now is that a lot of the members of my lab get together on Thursdays to go play volleyball in a nearby court after work! How cool is that?!? So naturally, I’m joining them!

Next Week on Martha’s Lab Adventures…

Adventures

Surprise, surprise– I got lost in the public transportation system again this week. You’d think I’d have gotten the hang of this by now… Instead, it took me two hours to get home last night instead of the standard one.

However, there are other things that I’ve gotten much better at, like moving worms from plate to plate with less civilian casualties, remembering to reserve space on the PCR machine before it’s all snatched up, and how not to burn myself on the bunsen burner that we use to sterilize the space that we’re working in.

I’ve done a lot of things right in the past week- I was beyond ecstatic when I ran the results of my vector cloning  on a gel to find that every single strand of DNA in the samples was exactly the right size and when I discovered that every worm I had randomly picked from the plate on which we crossed them was heterozygous for both traits that we’re looking at.

I’ve also done a lot of things wrong, though (oh, no, not another learning experience!)- like accidentally diluting proteinase K 33x instead of 20x because apparently I can’t do math (at all) or somehow messing up a step in PCR so that my genotyping of the second generation of my cross turns out a squiggly mess of none of the lines that we expect to see. None of these are irreparable, of course, but it can still feel like a major failure sometimes.

What I’ve learned this week is that in science, some things go amazingly (my vector cloning is still the pride of my life’s work to this point) and other things can go awfully (my genotyping of F2 worms), but life (and the experiment) goes on.

See you guys next week!!

Time flies

Hi everyone!

My grandma, who I’m staying with when I’m in Poland, always complains that I work so much that I’m never home. I’ve tried to explain to her that no one is forcing me to go to the lab early every morning or come home late, but that I like spending time there, learning about methods I’ve never heard of, and giving my worms baths (aka washing them from one plate to another when they eat all the bacteria on it).

It doesn’t seem possible that I’ve been here for almost two weeks already (?!?!). Two months seemed an impossible stretch of time when I came here, but judging how fast time is passing, it will be no time before I’m reunited with my family, friends, and most importantly, my dog!

Much of the things that I have been doing in the last week consists of moving worms from plate to plate, setting up a cross between two genetic mutants so eventually there will be progeny that lack both the functional NSPC group of proteins and the pqn-44 polymerase, and giving my worms a bleach bath so all the adults die and the eggs survive, hatching out worms that are perfectly similar in age–all things that make me feel sort of like an evil dictator with subjects who live to serve me. Now that these things are done, we can move on to more exciting things, like constructing a plasmid vector with mCherry (a fluorescent dye) to mark regions where NSPC-14 and NSPC-20 are active and infecting my worms with pathogenic bacteria to see the result that the lack of both NSPC and pqn-44 will do to the worm’s innate immunity and survival curve.

I’ll see you guys in another two blinks next week!

Martha

 

A Day of Firsts

Hi everyone, I hope you’re enjoying your first week without school!

Today was a day of firsts- first time using Polish public transportation by myself, first time going to my lab, first time working with C. elegans (which is actually much smaller than I expected it to be!) Naturally, I learned a lot. Some things that I learned today are:

  1. I have my very own project for the duration of my time here that my mentor trusts me with on my very first day! It’s concerning the Nematode Specific Protein C (NSPC) group of 20 proteins that are hypothesized to have a role in innate immunity, which I’m very excited about.
  2. The lab I’m working in is huge (more than 35 people!) and there’s no way I’m going to be able to remember the names of everyone that I was introduced to today.
  3. No matter how steady I think my hand is, the tool to pick up C. elegans worms (which looks like a straightened out, tiny, wire dental tool) shakes as if there was an earthquake happening at 4x magnification, making it very easy to kill or bury alive in agar the very worms that you’re trying to move to another plate.
  4. Lighting a bunsen burner with gloves on is not a good idea- my burned thumb still feels this one.
  5. Ordering the one thing that you can read in the scrawling handwriting on the menu is a safe bet.
  6. I need to pick a spelling of my name- Polish or American (Marta vs. Martha) before everyone gets confused about the labels on my boxes in the freezers.
  7. How not to sit at the wrong bus stop for 15 minutes before thinking “maybe it’s the wrong one.”

After today, I’m super excited to keep going back to my lab (with less public transportation derps).

I hope y’all are having as good of a day as I did!

Martha

Martha Kiela’s SRP Summary

Hello everyone!

I’m so excited to start my internship! I’m going to be working in a lab at the Institute of Biochemistry and Biophysics in Warsaw, Poland. A laymen’s summary of my project is basically that I will be trying to find out how a certain family of polymerases affects proteins that are important to the development of multiple myeloma, among other diseases. I know it has a lot of jargon, but here is a short and more exact summary of the project that I will be working on:

mRNA molecules are normally modified by the addition of a poly(A) tail by canonical poly(A) polymerases; however, recently, several new non-canonical polymerases have been found. Among these are the FAM46 family, which includes FAM46A, FAM46B, FAM46C, and FAM46D. The FAM46C gene in particular is mutated in about 10% of multiple myeloma patients, indicating its importance in multiple myeloma pathogenesis. In order to study the FAM46 family and specifically FAM46C, Caenorhabditis elegans is used as an alternative model. This species has a FAM46 homolog gene called pqn-44 whose function Vladyslava Liudkowska, the PhD student that I will be working with, is striving to characterize. To find the pathways through which pqn-44 is active, Vladyslava is working to create a transgenic line of C. elegans in which pqn-44 will be expressed with eGFP-tags. My project will be to work with her to identify these pathways by using various methods such as pull-down assays.

I can’t wait to start working in the lab!

Martha Kiela