A few months ago, my parents visited San Diego, and I brought them to see my work in the lab. Amidst the tour, I had to do a couple quick things for my research: streak out some bacteria onto a few plates, prepare a couple solutions for the next day, and the like. As they watched me pipet, their eyes went wide, and they tracked every move I made with a mixture of incredulousness and confusion. “Wow, you’re doing real science here,” they said. That made me smile. They didn’t know the half of it.
It was that day I realized that my family and friends really don’t know anything about what I do, what a day at work looks like for me. They just think I’m “doing science,” which has become an umbrella term that the whole world uses to describe what scientists do. This isn’t how it should be. My science is fundamentally different from the lab below me; while I study corals, they study ovarian cancer. The lab next to me studies regeneration in little worms, and the lab above studies bacteria that eat methane. The word science tells you nothing about what I do, why I do it, and what I spend most of my waking hours devoting my time and energy to.
In this article I’d like to tell you about what a day in the lab looks like for me, and what happens when I disappear into that big building for eight hours every day.
Wake up. Drink coffee. Extract sterivexes. Repeat.
I am currently at the tail end of a three-month research rotation in a lab led by Drs. Forest Rohwer and Linda Wegley-Kelly, two renowned scientists who study marine bacteria (among other things) at SDSU. PhD students usually spend their first year in grad school rotating through different labs, doing short projects that last a few months to see which lab might be a good fit for them to do their dissertation project, which can take up to 6 years (so you want to choose wisely). In this lab, I was given a project which involved extracting a thousand DNA samples from the demonic little filters you see to the right. The project would cost me my friends, my sanity, and the use of my left hand… (kidding, I still have all of these). But it did take a lot of time.
Over the past 15 or so years, the Rohwer-Kelly Lab has accumulated Sterivex filters from all over the world. This lab has helped the scientific community to realize over the past decade the massive impact that oceanic bacteria have on shaping, maintaining, and often damaging the marine environment. Small shifts in ocean conditions, like increases in temperature or pollution, can radically alter the communities of bacteria associated with an environment, causing disease, oxygen-starved zones, and other harmful effects.
Considering the oceans are changing at a rapid rate, the Rohwer-Kelly lab has made it its purpose to characterize these communities of microbes, and to get an idea of what they look like across the world’s oceans, in both healthy environments and degraded ones.
So, over the past decade and a half, students from the Rohwer-Kelly lab have gone far and wide: the Hawaiian islands, American Samoa, French Polynesia, the Caribbean, the Pacific coast, and more, equipped with SCUBA gear, (mostly) smiling faces, and their faithful tools: hundreds and hundreds of Sterivex filters.
The students collect water from all over the oceanic environment: from the surface waters, from the deep, and from water immediately above the reef, and using a syringe, they push the water through the Sterivexes. The filter inside is small enough to catch bacteria, which can be later extracted and sequenced to give us an idea of what the communities look like at that place and time.
Most of the students in the Rohwer lab have a horror story or two from traveling to such remote places, but for the most part, they love what they do. “Field work is the best part of science,” says Kevin Green, a recent graduate with a Master’s degree from the Rohwer lab. “But you need to go into it knowing exactly what you want to do, or else you risk becoming lost scientifically and never get a good story.”
Long days, little sleep, and hours spent diving also make field work one of the most challenging parts of science. Being on a ship for weeks on end, so far away from civilization can drive anyone a bit crazy. But even on our worst days, the samples still must be taken. Day in, day out, field scientists must continue to perform, to dive, to stay up filtering Sterivexes into the wee hours of the night. Field work isn’t for everyone. “But we are lucky,” Kevin affirms, '“we are lucky that we get to go to these remote reefs all over the world, and to experience places few people will ever get to see.”
With that part of their job done, the students quickly freeze the filters to preserve the bacterial DNA inside. When they return to the mainland, the Sterivex filters are promptly placed in the -80 degree freezer, where they are forgotten about for somewhere between 1 and 15 years. Then I came to town.
I showed up in the Rohwer-Kelly lab for my rotation with a smile and little bit too much enthusiasm. Intent on quashing that, my PI (the ‘principal investigator,’ or leader of the lab – aka, my boss), Forest Rohwer, was able to break through the ethereal mist keeping the Sterivexes hidden from memory in the -80 freezer. He proposed I lead the project of extracting and preparing the DNA from all of the Sterivexes in our possession, and that I do it in three months. Considering I didn’t even know what a Sterivex was yet, I wholeheartedly agreed. We called it the Thousand Sterivex Challenge.
(On a completely unrelated note, I have begun doing a little background research before committing to the projects that people ask me to take part in.)
And so I began extracting. Forest assigned me a team of 12 other students in the lab, and with their help, we extracted DNA from every Sterivex filter we could get our hands on in the lab. I extracted until my fingers were riddled with blisters and the sight of those damn filters made me want to put my head through a window.
exercising that pipettor’s thumb
The process is a not a complicated one, but it is long. It takes about two full days to extract 48 Sterivex samples, a little more if you include the other processes at the end. First, we thaw the Sterivexes, and attach little caps to the end so liquid can’t escape. We add some solutions to the filters, which are designed to break open the bacterial cells, releasing the DNA from its cellular prison. Following this, we incubate the filters in an oven at about 130 degrees overnight. This makes sure all the bacteria is off the filter and in solution, ready to be extracted and concentrated.
The next day, we add some more solutions to the filters to bind the DNA and make it play nice with our equipment. There’s a lot of junk in those filters as well: remnants of tissues from other organisms, the debris left over from breaking open the bacterial cells, and often sand or mud that found its way inside. We remove the stuff from inside of the Sterivex filter with a syringe, place it in a little tube, and spin it down inside a centrifuge to make sure that all the gross debris and stuff is stuck at the bottom.
We’re almost at the end, so bear with me. Next, we put the mixture of DNA and all those solutions I mentioned earlier into another little tube, but this one is equipped with a little filter in the middle: this filter is designed to catch DNA (and only DNA), and hold it there in place – this happens because DNA molecules are polar, like a magnet, so they stick to the polar filter really well. We put a few more solutions through this filter, most of which contain ethanol, which cleans the DNA and gets rid of the rest of the garbage in the sample, spinning the tube in the centrifuge each time. Finally, we add water to the filter, which, as a polar liquid, pulls the DNA off the filter and dissolves it. When we spin it one last time, the water/DNA solution passes through the filter and stays at the bottom of the tube: a clean, beautiful sample ready to be sequenced.
Whew. The Sterivex-extraction-two-day-marathon is over. But don’t get your hopes up too fast: we only did 50 samples. 950 more to go. Buckle up, the fun begins again tomorrow.
In the meantime, I spend my extra time making sure the samples came out okay, which involves checking every single sample on the Nanodrop, which measures the DNA concentration, and through 16S PCR, which tells us whether or not the DNA is actually from bacteria. Sometimes we get surprises, and sometimes we get no DNA. Such is the world of science. But for the most part, the process works pretty well.
many hands makes light work… or something
Three months of this flew by not entirely unpleasantly, busy as I was at DJing to keep spirits high, theorizing about the next season of Game of Thrones, and apologizing for involving the poor undergraduates in a project designed to crush my enthusiasm for life. It didn’t, by the way. I had a freakin’ blast.
On a serious note, this project was as exciting as it was challenging. When we sequence all of these samples, we will have an incredible resource with which to study the microbial communities in the ocean, an overwhelming amount of data with which to ask some really cool questions. We can study how the microbes differ in the same places over time (and across the progression of climate change), or how the communities differ in different places, and figure out why. We can see how the bacteria in polluted waters offshore of cities differs from water in clean areas, like the open ocean or over healthy reefs. We will be able to better understand the role microbes play in causing disease, and thereby how they fundamentally change ecosystems, such as they have done with coral reefs.
The list goes on. The data from the Thousand Sterivex Challenge will give us the tools to search for solutions to some really important ecological problems. It will give us a much better understanding of the world we live in, and how to make our impact on that world healthier and more sustainable. Knowledge is power, and we can’t hope to restore global environments if we don’t have a really good understanding of the stuff we can’t see, pulling strings from right below our noses and shaping the world around us. The Thousand Sterivex Challenge is a leap in the direction to change that. With that in mind, I would do it all over again.
This is what I did today. It’s what I’ll do tomorrow. It’s what I’ll keep doing until another project arises, and then I’ll do that. Research is moving from project to project, getting data – the puzzle pieces – and then stitching those pieces together into a more coherent understanding of the world around us. Scientists all over the world are doing it every day, and for the most part, we really like what we do. Next time you talk to a scientist, instead of lumping their work under the auspices of science, try asking what they do specifically. Chances are they’ll love to talk about it, and you may learn something on the way.