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.