Aware as I am that the inexplicable enthusiasm of the practitioner of any obscure hobby or field usually meets with raised eyebrows and questioning of one’s sanity, I thought I’d add a few interesting links that provide a broader context for the work I’m doing here at James Cook University. If you don’t believe me, take it from these authorities!
In the meantime, an update on my lab activities. As usual, I managed to shoot myself in the foot with one of the errors common among people who don’t do biotechnology for a living. I added loading gel instead of PCR ladder to my check, meaning that although I could see whether I managed to copy DNA yesterday, I couldn’t reliably say how big the piece I got was. Since the signal was weak, anyhow, we decided to re-run the DNA amplification PCR step a second time to double the yield of product before going through the clean-up steps.
In the meantime, I managed to complete the cutting and cleaning of the RpoB and RpoC gene copies I made, with a huge bit of help from the research assistant, Brendan. The images came through too late today to copy them to my jump drive for posting, but even a lay person will recognize the difference between the before- and after-images. Before today’s cleanup, there was a lot of extra genetic junk floating around. Now I have a nice clean sample, which includes only good copies of my gene. Tomorrow, I will run a ’sequencing reaction’ to mark the individual As, Ts, Cs and Gs of the gene sequences, and send them off to the sequencer for processing. With any luck, by next week, I will begin using ‘Sequencher’ software to align the genes from each plant to compare their species-specific genetic codes. I’m nearing the end of the beginning!
The last few days in the lab have finally begun to yield useful results, punctuated with what felt like many little false starts along the way. After a couple days away from the lab, I returned on Monday to find that I was all thumbs again, doing things mostly correct, which in a biotech context isn’t usually good enough. Each step along the way, I needed some patient correction to get to the next step of the process. Everything felt unnecessarily labored, and the weak results I got in my progress step check at the end of the day Monday left me feeling somewhat deflated. I returned Tuesday to the task, doing better, but still not moving along with the gracefulness and composure I’d like to have. There’s a certain kind of unflappable professional ethos called for by lab work, and I knew I was yet to find my comfort zone.
One of the problems has been the sheer number of process steps required by my project, each with its own little skills and techniques that require mastery. Because the equipment is a bit different than what I’ve used in my own lab work, my abilities are close, but not finely tuned to the work I’ve been doing here.
Resolved to make my mentors’ attentions, the sacrifice of my family and investment by my program yield a tangible result, I resolved to get into the lab extra early and get a head start on the setup required by the day’s work. After pouring a gel, I was certain that I’d bought a sizable head start, and felt pleased with myself as the lab staff arrived. Imagine my disappointment then, to realize an hour and several steps into the morning’s work, that I’d messed up even my simple preparatory step, which would require an hour to mitigate. Instead of being ahead, I immediately found myself behind. By midday, it was looking as if Wednesday would follow the humbling example set by the first two days of the week. As I finally loaded my extracted and amplified DNA samples into the tray, I half expected to get a poor result, and spend the rest of the day troubleshooting and re-doing my process.
The first break came when the results of the gel imaging came through. All of the samples of DNA I collected from plants not only extracted, but amplified nicely for most of the genes I’m studying. With the exception of two species for one of three loci run, I got strong results with only moderate background noise. The lab research assistant indicated that it was among the best such results he’d seen from seagrass DNA. Despite my pessimism, the yield of the effort was not merely a result, but a strong result. With renewed confidence, I was now on to the next stage of my efforts.
After collecting the plants, crushing and and extracting DNA from them, cleaning the DNA, making copies and separating it, the next step is to re-purify the genetic material from the gel. The mere act of grinding up a bit of leaf to get the cells open shears apart some of the DNA, so that small fragments end up mixed with the copies of the whole genes one is trying to study. All that DNA is blasted through a gel during electrophoresis to separate it by size, the way different diameter pebbles blast through a nested set of sieves. After doing this, the afternoon’s next step was to physically cut the correct ‘band’ of DNA from the gel, so that it may be prepared for ultimate sequencing.
To make it visible to work with, the DNA is marked with UV-sensitive chemicals that glow under black light illumination. However, since UV-light damages DNA (think skin cancer) a special setup is required to see the bands in the gel so they may be separated. A foil mask is placed over a UV light box, with a small window cut out so that a 1cm x .25cm area of light shines through. Using a scalpel, the gel bands are cut out carefully to avoid cross-contamination, minimize the excess gel taken, and maintain the correct numeric sequence. This all takes place in a darkroom, wearing a UV visor, gloves, and sometimes a backwards-facing lab coat to protect the neck from the light. Since one requires regular light to see where to place the excised sliver of gel, the entire process is a ballet of lights going on and off, blades diligently and accurately chopping, and piles of gelatin-like chaff accumulating at the corner of the bench.
For once, I managed to find a task I was good at on the first try, and I had processed the entire tray only 30 minutes later than the normal quitting time. I managed to get back 30 of the morning’s lost minutes, and come to a respectable point in the process.
Since all Wednesday’s work only provides information on one gene in each of the plants, I will repeat everything again with a second gene, with an additional set of time-consuming steps to prepare them for sequencing tomorrow. It’s double everything I did today, plus a bit more to do, in the same time I spent in today’s efforts. The challenge set before me (and my mentors) is to get two of the sample sets ready and off for sequencing by Friday, so that I may have a result available to me in time to analyze it with specialized computer software before I return to the States. My personal goal is to do one better and get not one but three sample sets for different genes away, so that I will have made a respectable contribution to the global initiative. With about 60 global species of seagrass, and 7 loci on each for barcoding under assessment, I hope to do 12 species for 3 loci in my time here. That works out to 9% of the international effort, notwithstanding necessary replications of results and the like. I intend to build on this work by taking my lab skills back to my students back home, and get up to the double digits! Will pipette for food!
My thoughts turn ever homeward, so it’s helpful to me to put my hands to work with something I can DO while I feel so helpless to do the part I want to for everyone who is depending on me. Maybe if I get this simple thing right, it will help play a part to open doors and make the lives of the people I care about a little bit better.
This weekend, I had several activities to do, related to my work at JCU, if not directly applicable to my sea grass project. Besides from continuing to work through readings and refreshing my understanding of the Hardy-Weinberg principle, we had a dinner meeting with local SRP-Australia supporters, a visit to the Reef HQ Aquarium and a trip to the Great Barrier Reef, 2.5 hours by boat off the coast of Townsville. All three were great fun. The Australian friends of the program are wonderful hosts, and they delight in injecting a social element into the academic purpose of our being here. The local aquarium has ties to JCU, and a member of the Heimann lab arranged visiting academic passes. The purpose of our visit was to see the ‘best practices,’ in marine ecology education at work. With the lesson guides and handouts they have published online in conjunction with the Great Barrier Reef Marine Protection Agency, teachers anywhere in the world have access to the materials they have prepared.
But however diverting these experiences were, the star of the weekend was the Sunday visit to the Great Barrier Reef along with a Spanish scientist studying reef fish. Her research uses genetic samples to study the population genetics of commercially important species, using many of the same techniques my project applies to sea grasses. The trip took 2.5 hours by boat, which battered the passengers on the outbound leg of the trip. Those lucky few who weren’t busy filling a colorful bucket with their morning’s breakfast caught a brief but fleeting sight of a humpback whale at a distance. But the high wind and waves made any but the briefest glimpse of the fluke impossible.
Once at the site, the crew moored the boat to prevent anchor damage to the reef. The location we visited, Wheeler Reef, is a protected as a part of the Great Barrier Reef World Heritage Site. We donned our equipment, and were quickly in the turquoise water, now becalmed by the platform of corals in the shallow water below the boat.
During my visit to the Reef HQ the day before, I mentally reminded myself that a showcase such as this was designed to delight visitors, and that in all likelihood, the actual reef would be more diffuse and less densely or dramatically populated by marine life. I was immediately overwhelmed with how wrong I was. The Reef was teeming with all manner of organisms, from giant clams to delicate tube worms. Large fish formed a silvery column under the stationary boat while sea cucumbers lay littered about the sandy sea floor. Clownfish wallowed in anemones, and shimmering blue chromis fish darted playfully in and out of the protection of a lavender tipped wreath of coral. The visibility was 20 meters, and we dove to an equal depth to reach the base of the reef before ascending into the lagoon of the cay, only 1 to 2 meters deep. On one pass, I noticed a green turtle emerge from below a shelf of coral, before swimming away into the blue void. I hyperventilated the first 50 bars of pressure from my tank before I adjusted to the sensory overload. Everywhere I looked, I saw something amazing which I could easily spend the rest of an afternoon exploring, were it not for the guidance of the master diver, leading me on a circuit of the reef. The only word I can think of to describe it is fantastical – something that a team of set designers or artists could only hope to approximate in their wildest imaginings. After returning via the mooring line with a superfluous safety stop and plenty of extra air in my tank, I boarded the boat, stunned by what I’d seen. I greedily ate a sandwich so that I could switch out my gear and prepare for the second dive of the day.
For my last tank, I sought to savor every moment underwater. The sense of the unreality of such a moment in one’s life was a constant companion. It’s hard to believe that one is in such a mythical place….that this is indeed part of the life you’ve lived forever after. I was relaxed and sipping the air I had previously gulped. I saw blue spotted stingrays and so many different corals, I lost count. There were fish I knew from pet store aquaria back home, and some whose beauty was a new revelation. Weaving in and out among the antlers and domes and shelves of coral was like visiting a cathedral of nature. Ornate in its exuberant forms, colors and sounds, it was easily the most remarkable natural place I have ever been.
The trip was half as choppy on the long way back to port, but I didn’t notice. The rest of the day, and the long bus ride back to school, I was riding on a cloud.
After Thursday’s success, there was genuine excitement to begin processing the samples collected at Shelly Beach. Combining the new samples with those recently brought back from Green Island and the Low Isles, the DNA barcoding project now includes a fairly comprehensive collection of the sea grasses of the Northern Queensland coast. But despite our enthusiasm, we first had to complete the dreaded move from the old lab space to the new one. For weeks it had loomed, but at last, years of old pressed plants, preserved specimens, old conference posters and broken down equipment had to be moved, cleaned, sorted, and stored or sent to the ‘tip.’
Not being much of an expert at anything, my job involved a lot of reaching high places, carrying heavy things, and taking trash down a couple flights of stairs to the dumpster. However, with all hands on deck, it proceeded more quickly and smoothly than had been expected. As a reward, we had a communal lunch, which was a wonderful opportunity to hear from the very busy heads of lab, and to get their first-hand accounts of research projects I’d only read about in my preparations for visiting JCU. I was pleased to get the inside scoop on what it’s like to fly aerial transects for dugongs off the Cape York Peninsula. I learned of the challenges of funding a sea turtle tracking program, and even heard prognostications of falling populations for herbivorous macrofauna, given the canary-in-a-mineshaft perspective our seagrass monitoring affords. A lot of people have given quizzical looks when I’ve talked about my work in this lab, but the longer I’m in it, the more I see the context it provides more glamorous work with sea mammals and reptiles. Without halophila, there would be no sirenians or green turtles or the like to speak of.
After lunch, I returned to my now-known form of documenting the collections from Shelly Beach. I pressed herbarium specimens, saved others in ethanol and took photographs of everything we’d found.
One pleasant surprise was discovering that the Zostera we had collected was in fact flowering, which provided an even rarer opportunity to take micrograph images of the sex organs of the plant. I was excited to go from complete unfamiliarity the day prior to being able to pick out individuals with sheathed anthers the very next day. Though wouldn’t count myself among the experts, my proximity to them makes the diffusion of their experience fast acting and efficient. I can learn a lot quickly when I have such authorities on hand to guide me.
Since my supervisor will be teaching this Fall, on Monday, our quest to get the DNA extracted from these individuals will again be thwarted. Instead, we’ll be in a lecture for advanced biology students. While I’m frustrated that the extraction will have to wait until the afternoon (meaning that PCR amplification won’t happen ’till Tuesday) I am excited to see what the class is like and to get the opportunity to have the “if I knew then what I know now” experience, since my own undergrad genetics performance was not my proudest academic moment!
After failing to retrieve useful samples of either Zostera or H. spinulosa on my trip to Magnetic Island, there was a flurry of discussion in the lab about where else these specimens may be collected locally. Since the tides are on an unusually low cycle, GPS waypoints that normally are further out in the channel are theoretically accessible from shore. With this in mind, and Doctor C’s Garmin unit in hand, we set out to take a second crack at these elusive species.
Pallarenda is a quiet beach-y suburb north of the Uni, marked by its wartime role as a gun emplacement point to protect troop and supply shipments between Townsville and the fierce fighting in Papua New Guinea. Now, it’s a national park and reserve for native flora and fauna. On a Thursday, the place was almost totally deserted. The car park is about two kilometers south of the last place anyone saw our seagrasses growing, so we started with a leisurely hike up the beach, timing our movements to arrive at the lowest possible tide. By the halfway point, however, we began to notice some unusual features of the site. the beach itself was a narrow strand between rocky cliffs and sea. Extending 600 m out into the channel was a broad, muddy flat, intermittently punctuated by patches of plant growth. Beyond, the substrate turned back into sand before plunging into the deeper waters separating the island from the mainland. Our target lay at the furthest point of the muddy span.
The closer we got, the deeper our legs sank into the muddy bottom, so our progress slowed to a crawl as we neared the 6 meter alarm. Once there, the Zostera wasn’t immediately evident. Exposed seagrass takes on a dried, blackish quality that challenges easy identification. Complicating matters was the inter-growth of four species on a common point. Using my hands like a large spatula, I worked my fingers below the dense mat of rhizomes, and lifted little by little an entire patch of grass. Only after I had the mass in hand could I begin to weed out (literally) the unwanted Halodule and ovalis. Besides Zostera, we were also rewarded with the palm-like H. spinulosa, that I’d managed to gather only a few sprigs of the day before.
We filled our bag with samples, and began the even slower, more exhausting slog back to the car. Slowed by fatigue, we began to notice things on the return trip we’d missed in our rush to beat the waves. A dead turtle on the beach. The osprey hunting the foamy strand. Millions of shells and egg casings and bone fragments of all sorts. It was enough to keep an army of beachcombers busy for life. It should be noted, however, that inevitably, all the best looking shells have a hermit crab living in them, and so must be thrown back. I believe that this is the malacologic corollary to Murphy’s law.
By the time we got back, we were caked in mud, and exhausted from the day in the sun. We filled the cooler and rinsed the best we could in the sea. I missed my dinner and was followed around Woolworth’s by a suspicious security guard, who must have taken my dazed and homeless appearance as indicative of my likelihood to shoplift. On the long nighttime bus ride back to the dorm, I was worn, but triumphant in having accomplished what I’d failed to the day before.
Today I joined the team of a different postdoc to piggy back along on their field collecting trip to Magnetic Island, located just off the coast from Townsville. They were undertaking many of the plant physiology experiments that I saw happening on Green Island. My task, besides providing heavy equipment lifting, was to seek out two specific types of seagrass previously sighted in the target area, including Halophila spinulosa and Zostera capricorni. The day looked promising when a small sprig of Zostera floated up to our boat as we set anchor.
Unfortunately, however, though I had luck finding many species we didn’t need (Halodule uninervis, Halophila ovalis, Thalassia hemprichii and Cymodocea serrulata) I was fairly stymied in locating my quarry. The first site was a small bay of the island, with waters 4-7 meters deep. Since it had been windy, there was a bit of a current, and the visibility, while good for the site, was only about 1.5 meters. My task was to swim at the surface, and free dive down to the bottom while holding my breath. If I found the plant, I was to collect it, but otherwise, I was to keep moving along. Closer in to shore, I hit a fringing reef, but the cover closer to the boat was quite patchy. After an hour and 30 to 40 free dives, I was chattering cold in the boat with nothing but specimens we’ve already worked with.
Since the divers were able to stay and work below without surfacing, on their second dive of the morning, they agreed to guide me to a spinulosa patch they had seen before. With new confidence and a relaxed breathing pattern, I returned to the water to do my own search while waiting for the signal. I became accustomed to my own zig-zagging path, catching my breath at the surface before making another ear-popping descent to scan a further seven or eight feet for my quarry. At the eventual signal, I raced to the spot and was thrilled to find spinulosa samples, but only a very few and scattered amongst the other species. I surfaced to catch my breath, but when I dove down, the current had carried me a few feet away, and completely disoriented me. With few landmarks, it was a constant challenge to get back to the spot where I saw something I wanted to collect, by which time I was out of breath and had to surface for air. By the end of the dives, I’d collected a few spinulosa, but certainly fewer than I’d hoped to sample.
With a record-setting low tide, we ate our sandwiches on the boat and relocated to an intertidal site that would be exposed by the unusually low waters. Here I was seeking Zostera, which favors muddy flats. But look as hard as I might, I found not one sprig of the elusive plant. I was pleased to be able to identify each of the other four or five species of the area by sight, but frustrated not to add to the pool of species in our DNA project. A random dog, whose tag read ‘Bunny’ must have felt sorry for me and joined me on my search. She seemed to belong to no one, despite her tags. She brought a charred stick that she’d drop at my feet, inviting me to play catch with her endlessly in the muddy reef flats. Everyone got a kick out of the dog who adopted me. I’ve never seen an animal so at home as she was today. She kept me company until it was time to return to Townsville at dusk.
Nothing is so exhausting as putting away a boat after a long day on the water. The rinsing, packing stowing, hauling connecting fueling, gate opening and closing and securing seems never to end when you’re wet and hungry and tired. Even the wild brush fires we saw licking orange flames and a column of smoke did little to stir me from the daze of a day of non-stop physical exertion. I slogged into the cafeteria as they were taking the trays off the steam table, still in my dive booties and smelly long sleeved shirt. I ate something as fast as I could, hopped into a long shower. I wrote a postcard to my wife, and went to bed. I have a feeling I’ll have no hard time falling asleep tonight.
Yesterday, after assembling the full seagrass team in the lab for the first time since my second day here, we paused to process the samples collected on the Low Isles before proceeding with any of the DNA sequencing. The idea is to get all of our extracted samples into a single batch, in order to save analysis time down the road. Interestingly, just before leaving, the divers were able hastily to retrieve samples of two less-well-documented species from our part of the world, Halophila tricostata and Halophila capricorni. The tricostata was easily distinguished from the rest of the pack, since its leaves are whorled and not shaped like any of the others we’ve looked at so far. The capricorni was tougher, since the defining feature is the absence or presence of fine hairs on the leaf surface. This meant each sample had to be carefully examined under a microscope before it could be identified. Getting the proper documentation took the better part of a day, cut across meal times, and left no time for DNA extraction.
Today, I finally had the opportunity to work a bit more independently in the lab, attempting to extract clean DNA samples from these 18 new seagrass specimens. Without the research assistant to guide me through his shortcuts, I took much longer to get through the many steps. Where two people finished in under a half of a day, it took me the entire day to get finished. Unfortunately, I think that the sluggish pace of the work may have dampened the results, because when we ran a check gel in the evening, the signal was pretty weak. This means I didn’t get much genetic material out of the plants. Fortunately, however, appears to be enough that we can adjust ratios in later steps to amp up the product to the same levels in our other runs. If this fails, we always have the backup dry, herbarium and alcohol-preserved specimens to work with, so even a failed effort here is not the end of the line. Even so, it was a humbling experience to focus as hard as I did, with a little experience under my belt, and get a less-than-stellar outcome. My interest is in developing a protocol which we can replicate in classroom conditions in New York, but I may need to find a more robust route to the same finish line.
Since JCU is on inter-session, the campus is relatively empty and quiet. This brief calm has lulled the animals from the hilly ranges around the Uni to come down and graze on the school’s lawns. Among the creatures preferring tender shoots of grass to the desert scrub of the hills are a certain mob of wallabies, which has been hanging around my dorm. There are three adults, one juvenile, and a pair of joeys. They’re fairly timid, and getting close causes them to stop their grazing, but I couldn’t resist trying to get a shot of the little one in its mother’s pouch.
After running the PCR reaction, a check of the results by gel electrophoresis indicates that for most of the samples, the reactions were successful in amplifying the DNA collected in the field. The dark bands on the light field on the image indicate the location of genetic material. The weak signal on the first region were re-checked with a second gel, and yielded the same result, despite the fact that it amplified for other regions. This tells us that although DNA was extracted, the mix of chemicals used failed to generate copies under the conditions applied. This means that we will adjust salt, temperature and reactant mixes until we are able to get a product that can be cleaned, sequenced and compared.
The last two days have been a sudoku-like challenge of trying not to confuse myself, or as they say here “stuff it up.” After confirming that I got DNA from all of the plants we collected, my next task was to dilute them such that each sample has roughly the same concentration of genetic material. Working with the image from the last post, I used Photoshop to approximate the intensity of the signal from each sample. Then I diluted each with water in a set of ratios to get roughly the same amount of DNA into each tube. Next, we had to mix up a set of three different cocktails, each with nine ingredients, to make the chemical combination required to amplify or copy the sections of DNA we are targeting in each plant. To someone more familiar with the process, I think it becomes habitual and intuitive. But for me, even with my lab background, I have to keep meticulous notes and must constantly tell myself what I’m doing as I go step by step, lest I commit a fatal error.
There are four different genetic regions being used internationally for the DNA barcoding of plants, and we’re throwing in a couple that have proven useful with seagrasses for good measure. In order to make sure not to mix up the identical-looking samples, I created an Excel spreadsheet that tracks each step of the process, as the numbers shuffle. They were first ordered by when we collected them, then by the amount of DNA in each, then by where they ended up on the tray. Practical necessities of working on the bench throw in an extra layer of variables, since there are so many tips on the pipette spaced so far apart, and only so many wells in the gel, spaced differently. The analogy that springs to mind is matching up 75 hot dogs (sold in packs of 10) and buns (sold in packs of eight) all cooked one of three different ways, except at the end of the day, one has to be able to say exactly which finished hot dog is made up of which bun from which bag, and which frank from which packet. By the time I’m ending, the sample number order is 2,6,1,3,4,5,8,9,10,11,13, etc. For the last step, I’ll have to un-jumble them all. To boot, the last four in each run won’t fit with their brethren on a single row of the gel, so the fourth row further mixes up the plot by slotting differently prepared samples in a common ’space permitting’ row. I had to make a map before hand, just so that I’d have a visualization of where it’s all going BEFORE I got downstairs to the lab.
Prior to leaving last night, we amplified the DNA using PCR (polymerase chain reaction.) This is basically a set of chemicals placed with the sample in an machine that gets hot and cold in a fixed cycle to make millions of copies of a stretch of DNA. Today, we will separate out the copies to see what (if any) results we achieved. If we have any luck, our next step will be to excise the isolated band of DNA from the gel, clean it chemically to purify the genetic material from other cell junk, and send it to a sequencer for processing.
As slow-going as its been, it’s still remarkable to think that a week ago, the material we’re working with was part of a piece of grass undulating peacefully under a clear Pacific sky. What took years now takes days, and if fortune favors us, we’ll soon have added to what the world knows about the species we collected.
