...and it's on to finals week!

While I don't have any finals myself this year, we've entered our penultimate full week of the semester and things are getting really hectic and will be even busier next week.

My schedule from now until the 21st or so will essentially be work at the bookstore and go into lab. The bookstore is going to start buying back books this week, so my hours will be increased dramatically to help deal with the influx of students. I'll also be helping out with our influx of books as they come in for next semester. Unfortunately, a lot of the professors haven't given us their book adoptions for the spring semester, so we may be super busy at the last minute putting all of the books out.

This week will be the first of many that involves unpacking heaps of books and finding their spot on the shelves for next semester.

I spent all afternoon and evening in lab on Sunday, trying to get caught up on some culture work that needed to be done. It's really frustrating when I put so much labor into something like a colony screen (to see whether a diatom culture has our GFP gene) and I get really unclear results with the PCR. I pretty much repeated this schedule last night (Monday) after all morning at the bookstore. It's starting to be crunch time!

And so it begins: lab all day on a Sunday.

I've come to the realization that I don't need to sprint to the finish line with my lab work this semester. I sometimes forget I have more time than I give myself to get something done. All I really need to do over the next two weeks is put together a powerpoint for my lab project and give a presentation next week. I hope to get some good writing done in and around that in addition to some solid lab work.

Ehhhh...

Off to start my Tuesday.

It was 57°F and sunny on Monday, and I missed almost all of it while I worked at the bookstore before going into lab.

Thursday Science

Colorful labels mark primers for my real time-PCR reactions.

Setting up said real time-PCR reactions next to the special PCR machine that will quantify my cDNA of interest.

The computer screen displaying the real time-PCR software we use.

Going to do a bunch more RT-PCR tomorrow. Wish me luck :-\

From GRE to RT-PCR

The view of Lasry the other day on my way into lab.

A few weeks ago I took the GRE as part of my quest to continue graduate school in a Ph.D. program. Standardized testing is such a money scam given all of the fees involved from taking the exam to getting your scores and sending them to your schools. Furthermore, I don't think standardized exams are particularly informative, which is funny because Clark was just in the news because they are no longer requiring SAT scores for undergraduate admissions.

But I digress: I'm not here to complain how ridiculous the whole process of taking the GRE is. I'm just super glad the GRE is behind me and I can focus on things that actually matter. Yet ever since, I've been taking it slow with my workload because I needed a bit of a break. Thanksgiving marked the end of that break though, and now it's time to jump right back into things to finish up a productive semester.

I have a very hopeful list of things I'd like to accomplish by the end of the semester in mid December. But because I'll need to be working a lot more hours at the bookstore to help with the end of semester rush, it's going to be a big squeeze.

We're starting to get books for the spring semester at bookstore, & they're piling up.

This  picture is enhanced with the iPhone app Cat Effects.

Meanwhile in the lab, I have heaps to do. I want to finish up my experimental assays, screen and grow out more diatom cultures for future experiments, prepare a presentation and do some writing, and finally start with some RT-PCR experiments.

Now previously I extracted RNA from my frozen cell samples and used the mRNA present from the cell samples to generate cDNA.

RT-PCR, or real time PCR, will allow me to quantify the cDNA that I have made from my RNA. This is because RT-PCR can measure the number of DNA copies at each copy cycle by measuring the fluorescence of a special dye that hybridizes with the DNA. This is pretty snazzy, but it looks like I'm getting myself into a lot of grunt work.

You see, in order to get precise data from RT-PCR, one usually runs three replicates from one DNA sample and compares the output. But it's not like I only have one DNA sample. I have tons.

This is because I have 8 DNA samples at the minimum per experimental assay that I run in the beginning: I have two different cell lines that I test per assay, which get divided between two different test conditions, from which I take cell samples at multiple timepoints.

For the time being, I'm going to start with time zero and my end time of 60 minutes. This will hopefully give us start and end data that will display the overall trend of my experiments.

But because of positional effects, I need to run more than one experimental assay. I'll probably run three sets of samples through RT-PCR, but I hope to have up to 5 samples completed and ready for RT-PCR by the end of next week.

This is from today while I waited for my next sampling time.

Wait, what's that positional effects you just mentioned a second ago? Oh, yeah, positional effect. Because we transformed our diatoms with ballistics, our DNA was randomly inserted into the diatom's genome. By chance, the DNA we're trying to measure in our experiments might be inserted into a region that is either expressed more or less often, which would skew our data. By gathering data from multiple replicates, we'll get more accurate data regarding gene expression. Cool how science works, huh? We think of the neatest little caveats. Well, I mean, my professor does.

But sometimes I do too!

Like last month or so I came up with a solution as to why our cultures weren't growing so well. And then more recently I figured out how to screen my diatoms for our reporter gene more effectively.

So yes, sometimes I come up with cool things too.

The Jonas Clark building. I can't stop taking pictures from this vantage point! Gah! I love it! 11/18/2011

Marine Biology Research

I made a new video discussing what it's like to do research at Clark. This video pairs really well with the video I made called Bio @ Clark.


Marine biology research at Clark University.

Extracting RNA from DIE-atoms, among other things

I've been very busy in lab and such, but managed to post a new video onto the YouTubes, which you can see right here:

  
Warning: awesomely awesome intro in this video that's totally worth watching.

It's funny, we had a pretty cold and slightly miserable October that brought us our first snowstorm of the year, but so far November has been very kind to us. Yeah, today it was cold and rainy, but we've had temperatures in the upper 50s and low 60s for most of the month with plenty of sunshine.

While I've enjoyed the beautiful weather on campus, I know all too well it'll get really cold once again and more snow will fall. But until then I'll be sure to appreciate the nice fall weather as long as it lasts.

October Snow Storm Carnage

Last night on October 29th, central Massachusetts got a fair amount of wet snow. Estimates and measurements vary, but the campus got about 5-6" of snow. The problem with wet snow is that it weighs down trees, especially if the trees still haven't lost all of their leaves yet. The problem is amplified with longer branches as it creates more downward force on the branch (basic physics thank you), causing the branches to snap off.

I've taken a bunch of photographs and have posted two videos, here and here. I'll also embed the main video at the bottom of this post.

A somewhat complete photo album is here.

My tumblr post is here.

A slideshow on the New York Times of some of the damage to the northeast.

Before the storm: Saturday October 28th, 2:30 PM.

After the storm: Sunday October 29th, 12:00 PM.

My favorite post-apocalypse photo.

A sample of the carnage:

A video of the tree carnage on the Clark campus.

Measuring gene activity in changing environmental conditions

Yeah, so, I'm ready for science.

If I do say so myself, I do look rather badass in this clip. Honestly thought, scientists should be looked up to like super heroes because we're going to be the ones saving the planet (hopefully). More about this topic another day.

Anyway, I've started the phase of my research project that will yield be data. Data for my thesis. Yesssss.

Although my last few videos have been quite long, this one below is short and sweet. How awesome.




My tumblr graphic representing the video >_>


While I still need to plan out all of my assays and assay replicates (to strengthen the power of our collected data), the fact I'm starting this process has me really fired up. For the most part, I'll be running cultures for an hour or so under continuous light (because diatoms like light) under different environmental conditions and taking cell samples periodically for later analysis.

But really, I should be studying for the GRE right now because I take it in less than 3 weeks.

In other brief news:

Last week when I was about to load a gel, I noticed I had a friend. (It's a spider. Click the link!)

In my spare time I put together this Hawk vs. Squirrel video that captures the relationship between the Clark squirrels and the area hawks.

I also updated my Fall @ Clark section, a collection of fall photographs. My most recent additions are at the bottom of the page.

Diatom Colony Screen

A new lab video!



In this video I discuss designing a colony screen protocol for my transformed lines of diatoms. In one of my recent posts, I posted this picture diagram which oversimplified the process:

While the PCR steps are the same (starting in the top left panel with the diatoms being combined with the PCR mix), I discovered that diatoms grown in liquid culture (i.e. their natural state) needed extra care in preparing them for a colony screen.

Let's step back a bit and quickly talk about colony screens (in case you didn't read my above Tumblr post). A colony screen is a modified PCR reaction*, which is a cyclical amplification of a short sequence of DNA, exponentially copying the targeted DNA strand. (*Even though PCR stands for polymerase chain reaction, PCR is usually said out loud in conversation as PCR reaction.) Often the DNA source for a PCR reaction is a purified, such as plasmid DNA purified by a process to separate plasmid DNA from proteins and genomic DNA.

But colony screens use a colony of cells to supply the DNA for the PCR reaction. By initially lysing the cell by cooking it at a high temperature for a period of time, the cell's DNA is released into the PCR reaction mix and then amplified in the reaction. Colony screens are used to screen cells for the presence of DNA--that is if the targeted sequence of DNA is present, it will be amplified by the reaction. If a reaction gives a positive band on a gel (bottom right of the colony screen chart), then we know that the DNA from the cells in that particular reaction also have the DNA of interest.

This colony screen method should in theory work for other cells, like diatoms. My initial trial successfully amplified GFP from diatom colonies growing on agar plates, but it didn't work for diatom cultures growing in liquid media, a distinction I didn't make at the time.

I soon realized after a failed trial of screening  diatoms solely from liquid cultures that the residual salt water from their growth media was throwing off the delicate chemistry of the PCR reaction. This graphic paired with the above video discusses just this.

Fall is creeping around the corner!

...now only if this rain would finally go away so we could enjoy this early fall weather...

Untitled post about science

Science on a quiet Sunday afternoon:

Post-transcriptional regulation: modulating protein synthesis

In other news, this is a poster I made for Tumblr advertising my blog. I likes it.

Biology... to be continued?

This completes my second Biology Ph.D. program application. *Crosses fingers*

Recent collection of artfully displayed pixels

I've been piling up cool pictures both from lab and campus and wanted to share them with you. They're all in pretty high resolution, so please click on them to see them in their proper, large form.

40 glass culture tubes wait to be inoculated with single colonies of transformed diatoms. Each tape color represents a different gene cassette that was transformed into the diatoms.

A few days ago, my friend Miguel set up an "intertidal" zone in the lobby of the bio building for the Marine Biology class.

A recent panorama of the green on a busy late summer afternoon.

One of the craziest sunsets I've seen in Worcester--taken just two days ago.

I love clouds, especially these ones.

Again, one of the best Worcester sunsets I've seen in ages. This was taken in northern Worcester, about 10 minutes from campus.

Another cool sunset seen from my porch.

Fluorescence Microscopy, Cricket, stART, & Gene Specific Primers

I uploaded a new video to YouTube this weekend (posted below) chronicling my latest efforts in lab and a few things on life around Clark.

This video touches on:

• how I'm trying to use fluorescence microscopy to determine which cultures of my transformed diatoms possess our experimental plasmids that express GFP
• the Clark international students playing cricket on campus
• the annual Worcester art in the street festival (stART), held along Park Avenue

Since this video was recorded, I've made further steps in my project. In order to test cultures of diatoms sooner for their presence of GFP and at a much higher volume, I've been designing gene specific primers to amplify regions of DNA in the diatoms, that would yield bands of DNA if the GFP is present. There are four main sets of primers I've been designing and will use in PCR experiments:

By using the standard NR-eGFP-NR construct as an example, the sets of primers would like the above picture, where orange highlights show where primers would amplify if the sequence exists in the DNA. Primer (1) would yield segment [a]: a portion of the 5' UTR, eGFP (enhanced GFP), and a portion of the 3' UTR. Primer (2) would yield segment [b], a portion of the 5' UTR and a portion of eGFP; primer (3) would yield a segment [c], portion of eGFP and a portion of the 3' UTR. Primer pairs to amplify the (1, 2, & 3) regions will be made for all of the gene constructs, that is NR-eGFP-NR, NR-eGFP-Actin, NiR-eGFP-NiR, and NiR-eGFP-Actin. Therefore only a specific set of primers will be used per diatom culture, depending on which plasmid it is supposed to have.

All of the plasmid however will be amplified with the fourth and final primer pair. Primer (4) would yield an eGFP segment [d] if the diatom cells had it. This will be the most important primer result out of all of them.

Once we have these primers at hand, I can screen many colonies at once. All I need to do is take a small DNA sample from a colony, add it to a PCR reaction, and run the reaction. I should be able to quickly decipher which colonies have GFP based on an electrophoretic gel.

The Genetic Transformation of Diatoms with Inducible Expression Plasmids

Last month my undergraduate/now graduate research project at Clark University reached a new level of awesome with the transformation of diatom cells with plasmids I've been working on for several semesters. It's been a long time coming for sure, with both problems encountered in the lab project and juggling other college courses.

In my blog I've chronicled some steps along the way on my project, and I've talked about:

• phase one of my project and phase two of my project and creating posters to present my project at campus-wide functions,
• transforming bacteria to clone pieces of plasmid and constructed plasmids,  
• cutting pieces of DNA out of plasmids so they can be pasted into different plasmids,
• sequencing pieces of my plasmids, 
• constructing my plasmids,   
• completing my NiR plasmids
• and preparing for the diatom transformations.

And now I can talk about the next step of my project: the transformation of diatoms with the plasmids I've created.

Now I am undergoing the process of selecting lines of transformed diatoms for the next step in our project. But we've recently realized that the light intensity in our growth chamber is much brighter than previously published experiments growing transformed cultures of diatoms. Increasing light intensity reduces the amount of chlorophyll made by plants and algae. Why is this important? Two reasons:

1. Less chlorophyll will reduce the pigmentation of our cells, making them harder to see once we transfer them to liquid culture.
2. Our gene giving resistance to our antibiotic (that allows us to select for positive transformants) is driven by a chlorophyll-associated protein, which means expression may be reduced in higher light intensities. If chlorophyll expression is reduced by high light levels, then this may reduce the activity of the chlorophyll-associated protein that drives the antibiotic resistance in our diatoms, which means less resistance to the antibiotic in the media. This ultimately means reduced or no growth.

Now, our diatom cultures have been growing okay on their agar plates, but I'm beginning to wonder if they're growing slower in liquid cultures, where they're more likely to absorb more light. To combat this, I'm growing some liquid cultures behind layers of porch screen to reduce light levels penetrating the liquid cultures. In the below picture, you can see the different diatom cultures I have growing right now:

Transformed diatoms grow on agar plates (left foreground) an in liquid media (left background and right).

I've been growing diatoms in 3 mL cultures in little dishes, which you can see in the back left. Groups of 3 dishes sit in a petri dish to allow easier transportation. These dishes however require 3 mL of culture just to cover the entire bottom of the dish, which means the cells are going to be fairly diluted. I also started 1 mL cultures in the test tubes on the right, where porch screen blocks out a lot of the incoming light. Some of the 3 mL cultures also have screen on top of them to block out some of the light.

Aside from trying to grow our cells in liquid culture, I've been looking at them under a fluorescent microscope to try to determine whether our cells are fluorescing due to GFP expression or whether it's residual glow from chlorophyll. UV light from the microscope excites all pigments in the sample, and then I look at the sample through different filters that only allow certain wavelengths of light to show through. However, our current filter set up hasn't allowed a clear delineation between GFP and chlorophyll yet. Most of our pictures look something like this:

...but they usually have a lot more background color in there too.

My next steps will be to either design or order commercial GFP GSPs--gene specific primers for GFP. That way we can run a PCR on some diatom DNA and determine whether they have the GFP gene they are supposed to be transformed with. Because I have a great control (the original plasmid DNA), I can test a whole bunch of diatom colonies at once and be able to select appropriate lines of diatoms faster.

Glowing Green Cells

Just before the weekend, my professor wanted to look under the microscope to see whether our transformed diatom cells were expressing our reporter gene GFP. I've been culturing several different lines of transformed diatoms since the transformation. These cells were co-transformed, which means the resistance plasmid is separate from the GFP plasmid--that is the transformation process shot two different plasmids at the diatom cultures. Therefore we need to select for diatom lines that posses both plasmids: the resistance plasmid and the GFP plasmid.

Below is a poster I made briefly describing each of the three main steps in the process: transformation, growing the diatoms on selective agar plates, and subculturing diatom cells in selective liquid cultures.

The diatoms will only grow if they received a copy of the resistance plasmid. And well, we can tell that the diatoms don't particularly like the antibiotic we put in the growth media:

By performing a simple antibiotic sensitivity test, we can show that diatoms will not survive in the presence of the antibiotic unless they receive the plasmid that confers resistance.

Below is the first round of pictures of cells expressing GFP. These pictures came from a single colony on an agar plate, which means I didn't transfer it to a liquid culture (i.e. it was sacrificed for science). However it does give us promise that we will have GFP-expressing lines of diatoms in the near future/ I've been working on growing up the cells in liquid cultures and hope to have something by the end of the week. After that, it's a matter of growing up larger liquid cultures before we can test some science!

The view through the microscope. So much green!

This frame shows some of the variability in the intensities of fluorescence. Denser cell populations will appear brighter.

Some clumps of cells are larger than others.

The start of the semester & culturing transformed diatoms

The backside of Goddard Library, one of my favorite photo spots.

The Fall semester has quickly begun at Clark University. While I was worried that Hurricane Irene was going to make move in day for the sophomores, juniors, and seniors messy, Irene barely inflicted any serious weather upon Worcester (we were lucky). I've been working at the bookstore the past two weeks where it has been very busy, as students have been scrambling to get all of their books on time. As a result, I kind of missed the first week of craziness on campus for better or for worse. But I always love the start of the semester--everyone is always so happy to see each other and be back on campus.

On Friday, we had our annual student activities fair, where almost every social club on campus from CUFS (Clark U Film Society, which shows movies on Tuesdays and Thursdays in our cinema) to ROCU (Radio of Clark U) is represented by student members. This is a great way for incoming first years to discover all of what Clark has to offer for our ever expanding extracurricular groups.

A panorama of the student activities fair, as seen from Main St.

The fair from the window of Tilton Hall, nearly showing the fair in its entirety.

Additionally, I posted a new video on YouTube chronicling the start of the semester, Hurricane Irene in Worcester, and last but not least, my most recent lab work:

Everybody's working for the weekend (or just grad school application money)

Recently, I've pretty much only had time for two things: working at the Clark bookstore and looking into grad schools. Because the bookstore is crazy busy the first few weeks or so, I've been working more than twice as much as I'd like to in order to help out. I sincerely hope things start to slow down really soon, because I've been working a lot the past two weeks. While this means I'll have money (which I can then spend on grad school applications), I've had little time to do anything else.

Coincidentally however, this is a perfect time to be busy at the bookstore when it comes to lab work because I'm in the process of growing my transformed diatoms on selective plates, a process that is going to take me at least two weeks before the next step. This next step may come as soon as tomorrow, before another one to two week growth period before I can even start to test my diatoms. But this isn't the topic of this post.

In my spare time when I'm not feeling completely exhausted from my tiring shifts at the bookstore, I've been looking at Ph.D. programs to start after I finish my Masters project here at Clark. This is actually a much scarier topic than I've lead myself to believe, because it doesn't take into account a lot of unknowns, such as whether I'll be able to get into grad school, whether I'll be able to finish my Masters project on time, and whether my girlfriend and I can get into grad schools remotely close to one another. While a lot of this is out of my control at this point and time, it doesn't make me feel better when I think about it. The best I can do at my point is get a first round of applications done as soon as possible so I can apply to my top schools as soon as possible. Applying to Ph.D. programs early is important, because there is only so much money at each school to support students. While I have to work at the bookstore again this weekend for an afternoon and I have some other things to clean up, I hope to have my act together on my first round of schools by early next week, and have contacted my the professors I'd like to work with the most.

It's embarrassing to admit, but I haven't even taken the GREs yet and I won't take them until mid-November, only a few weeks until some applications are due. Depending on how my applications are, I may have to bite the bullet and pay the stupid fee and bump up my exam day. Originally I was worried I was going to be weighed down with applications and lab work too much in order to properly study for the GREs. While that still may be the case, I'm wondering whether taking the GREs earlier would be better, so I can have a complete package application sooner.

That reminds me... I need to contact two more professors about writing me recommendations. Oh boy. This is what happens when my schedule becomes abnormal for two weeks thanks to working at a "real" job--I lose track of things. Along the same vein, it's weird how I feel like I've missed the start of school because I've been stuck at the bookstore the entire first week of school. *sigh* I've even started to panic that I'll miss the best weeks of fall, but I'll make sure that doesn't happen. I'm really hoping the bookstore will start to slow down really soon (as early as the middle of next week), so I can work less and get my act together in lab when the need arises.

Lab Photos

I've been taking a lot of photographs in lab to post on Tumblr, as a quick way to share my work in the lab. Here is a recent collection of my favorites.

My bacteria have a habit of spelling out scientific messages, but this time they took it to a new level!

A plasmid DNA resuspension. Black permanent marker outlines the pelleted DNA from an ethanol precipitation at the bottom of three tubes, a trick I’ve started to use in order to aid the resuspension of DNA into a smaller, more concentrated volume. The outline allows me to precisely add a very small amount of buffer to the pelleted DNA, as it sits taped at an angle to a pipet tip box.

After completing a set of inducible expression plasmids which I transformed cultures of diatoms with this past week, it was time to throw out several months worth of agar plates. These agar plates were home to different lines of E. coli which I used to clone different parts of my plasmids. Now that I have glycerol stocks of these E. coli lines (frozen stocks of bacteria cultures stored at -80°C), I was able to clean out my bin of agar plates. Not only did it feel good to get rid of so many plates, but this is an exciting stage in my project as it starts in a new direction.

Genetically transformed diatoms scatter the surface of a selective agar plate. The agar plate, specially formulated to support the growth of the microscopic algae, is made with the antibiotic ClonNAT which is strong enough to prevent the growth of diatoms without a resistance plasmid. The genetic transformation was a co-transformation of a pair of plasmids: the inducible expression plasmid which we will use for genetic regulation tests, and a resistance plasmid for ClonNAT resistance.

This is a new “invention” I came up with the other day in lab: a simple pen/marker holder. While it’s only big enough for 2-3 markers, it’s perfect for our lab benches because our permanent markers wander off. This way our markers stay put on one side of the lab bench. I used clean (but previously used) 50mL conical tubes with a bit of lab tape.