Mind the Gap

My little FringeFrivolous Blogging UnConference came and went this past Friday, and by all accounts a good time was had by all. Several minor miracles transpired:

• it did not rain on the roof terrace
• everyone had a place to sit

• not one of the fifty-odd (or should that be fifty odd?) delegates plummeted precipitously to the Farringdon sidewalks below

and, most importantly,

• we did not run out of booze

The two winning UnTopics also had the best titles: LOLcats and Labrats and Science PR: do we need a Trinny and Susannah for scientists?. The group conversation was energetic and enthusiastic – there are some great photos from Victor here, and Richard is just putting the finishing touches on a super video montage now published on LabLit. As for me, I came away with a lot of good blogging ideas for the future.

This post, unfortunately, is not one of them. My spirits have been completely sapped by a fairly unrelenting work schedule of late, and when I found myself weeping over my test tubes for no reason and almost getting into a fistfight over a misunderstood latté order at Starbucks, I realized that it was time for a vacation. So from tomorrow I’m off to swim in the sea in Kent, do a bit of rambling and then spend about a week at home finishing my third novel (only fifty or so pages to go!).

Play nice, all.

Sometimes it feels a little bit too good to spend money in the lab.

Blue streak If you have to ask, you can’t afford it

For those of you following the story so far, I recently triumphed in my new career as a Cloner For Hire. In brief, I managed to make and verify three complex DNA constructs, finishing the very day my visa-frazzled colleague returned to London to pack for his sabbatical. This had already turned out to be a bit more involved than I had initially anticipated – and then the boss suggested I extend the favor by recombining the little blighters into baculovirus vectors (so-called ‘bacmids’) so that my colleague could furiously finish up a few final experiments for the paper in the week before his departure.

The bacmid kit seemed straightforward, but I did notice that the transformation step – the part of the procedure when you zap bacterial plasmids into E. coli and allow them to recombine with a resident bit of DNA – looked a bit problematic. I could tell something was up by the cagy way that the kit’s instruction manual phrased certain things. That the procedure was far from efficient was also clear by the modifications in the standard protocol: five hours of growth before plating onto the triple antibiotics plates instead of thirty minutes; two days’ growth after plating instead of one; and then a further re-streak of ten prospective white colonies to confirm that they actually were bona fide recombinants.

At first I thought the company was just trying it on when they suggested purchasing their own special – and highly expensive – derivative of X-gal, the chemical substrate you need to put into your agar plates so that you can tell which bacterial colonies have successfully recombined (blue colonies are unperturbed, whereas white colonies are bacteria that have lost the beta-galactosidase gene because the genetic modification of interest has disrupted it). But then I thought about it: two days to get color development and a re-streak to confirm it? These colonies were likely to be pretty anemic, and even freshly prepared X-gal can often result in very pale colonies. We simply didn’t have a day to lose on ambiguity.

Dear reader, I bought that expensive X-gal derivative. And I have never seen such lovely, deep-blue, almost slate-purple colonies in my life. I thought I would weep with joy, and my colleague as well – it was KimWipes all around. And it’s a good thing I did too, because after re-streaking the white colonies, the blue controls were so faint the next morning that I spent many, many minutes squinting at the colonies, trying to decide if there was the faintest whiff of blue about their whiteness.

The moment of truth came in the form of PCR confirmation. There is a wonderful moment in science just before a result, no matter how routine. For me, even after two decades in the lab, I still get a frisson of anticipation when an agarose gel is sitting on the lightbox, the verdict unclear in that hushed moment just before the UV source is switched on:

And then –

– you see that your bands of DNA spell out success. It’s a priceless feeling.

Just an hour before the Fedex deadline, I was coaxing my final bacmids into solution and labeling up the tubes to ship them off to my colleague’s sabbatical lab; with a prevailing wind, they’ll rock up at about the same time tomorrow morning as he does.

Time, I think, to saddle up and head off into the sunset.

With advancing years, I find myself taking on the annoying traits of the older people I used to secretly pity. Yes, the young really don’t know how good they have it, and no, the world would not actually be a better place if we all spent every waking hour wearing tie-dyed T-shirts, protesting about injustice and trying to save the whales.

But the opposite seems to be happening to me in my scientific life. When I was just starting out in research, I was fascinated by the rise of kits – those experiment-by-number packs which must surely represent the ultimate lure of scientific youth. The lab in which I did my Ph.D. research wasn’t prone to frivolous spending, but I would watch colleagues in richer labs with envy. These kits never opened doors to techniques that would otherwise not be possible: they just made things easier. It was the same reactions, packaged up in neatly nested bottles and tubes with a handy instruction manual. Typically, the kits involved some pretty major short-cuts, were often less toxic and, probably most admirably, freed you from the tedium of having make all the solutions yourself. Scandalously expensive, they represented all that our betters were constantly warning us against: mental laxness, fiscal extravagance and (the unuttered subtext) the worst possible species of moral weakness. What sort of real scientist (we were told) would spend $300 on a kit when he could cobble together the necessary chemistry for a fiver?

Urban myths started to circulate, too, near the end of my Ph.D., about candidates caught out in their oral exams when being asked to explain exactly what Solution A did to Substrate B in that shiny blue box. Today’s youth, we heard, had become mindless drones, performing manipulations without truly understanding what they were doing. All of science would surely shudder to a halt.

But, you know what? That never happened. The kits persisted, and increasing numbers of labs started to use them. Time is also money, and what is a better use of time, spending two hours fiddling with an analytical balance and a pH meter, or doing a real experiment with a reagent you prepped in half the time using a kit? I certainly don’t miss the toxicity: as someone whose trendy 1980’s jeans are probably still quarantined in a radioactive landfill somewhere near Bethesda, and who once spent a memorable couple of hours in a Seattle ER getting phenol rinsed from her eyes, I appreciate the clean, safe workflow of modern luminescent detection systems and nucleic acid-binding columns. And as someone who once wasted four months of her life because a technician inadvertently messed up the recipe for a simple buffer, I like the reproducibility and reliability of the pre-made solutions. I still do a lot of stuff the old-fashioned way, but I’m increasingly succumbing to the allure of ease that kits provide. I know they are more expensive, but I do wonder how much money they save in the long run.

At the moment, I’m elbow deep in a wondrous new kit. After successfully completing the three constructs I promised to make for a colleague, I’ve got a week left to recombine them into insect virus ‘bacmids’. I may have a Ph.D. in retrovirology, but I don’t know my baculoviruses from my backside. With the kit, I don’t have to. Yes, I’m not ashamed to admit that I’m just following the instructions line by line without having the slightest idea of the biology that will transform my miniprep DNA into one of these do-hickeys capable of creating transgenic insect virus particles. If it all goes to plan, it doesn’t matter. (I feel deliciously naughty just admitting that in public.)

So that’s me: the aging kit convert. I think I’m just too old to do molecular biology in a cardboard box in the middle of the road like my grandma used to. Is that so wrong?

In the true spirit of spontaneity and general dishevelment of my life at the moment, I hereby announce that our 21st August event will not take place at the RI as previously announced.

Instead, we’ll be gathering at the offices of Mendeley. Props to Victor Henning for arranging not only a gorgeous roof terrace, but also Mendeley’s generous sponsorship towards drinks and snacks on the night. (I’m not sure he can secure summer weather to go along with it, but fingers crossed. In case of rain, never fear: I am reliably informed that the indoors bit is rather swanky too.)

The address is 9 Back Hill, London, in the Farringdon area, and you can see a map here.

Ideas for Untopics are already being exchanged on our dedicated forums – do register your interest and join in the brainstorming session if you plan on attending!

Are you a blogger, a former recovering blogger – or just blog-curious? Do you live in the London area or plan to be in town for the Science Online conference? Would you like the chance to meet up with your fellow addicts for a wee libation or three on Friday the 21st of August?

I’m organizing an informal evening get-together for bloggers (and interested hangers-on) on 21st August. From 7 PM, we will invade the Royal Institution’s hallowed (and alarmingly pink) café/bar area and make some noise. My spies inform me that a number of bloggers are up for this, so we expect a good turnout. For those of you taking the guided tour with Matt Brown, he has kindly volunteered to herd any willing victims along to the venue – provided he doesn’t lose you down some crevasse. If there is enough interest and people, we might even take a stab at an ad hoc Fringe-Frivolous Unconference, as the topic of blogging itself isn’t much on the SoLo agenda this year, and not everyone could make the North Carolina conference. But at the very least, we will have a chance to socialize, drink, and otherwise be frivolous — and I’ll organize a few flip charts just in case.

And if we’re very, very lucky, it won’t degenerate into karaoke.

Please register interest on the comments thread – both about attending, and about whether you’d be up for an impromptu meta-blog Unconference. And please do spread the word!

Glancing at the calendar this morning, in these holiday-stagnant, seminarless summer weeks in the lab, I realized that I am coming up to the midway point of my current research appointment. Like that strange illusion of endless summer that most American kids probably experience when school breaks for three months in June, January 2012 seems like a long way off to me. For me, here, I’m still a child in July: the summer birds yet linger, the goldenrod has not yet started to bloom and the evenings continue to glow right up to bedtime.

Today, I exist as a pure research being: setting my own agenda, following the science where it leads. I have a salary, a secure semi-independent affiliation, a generous consumables budget and no obligation to write grants. But sure as the leaves will turn gold, the geese will fly and the mornings will become edged with that perfect crisp coldness, this dreamy period is bound to end. And when the metaphorical autumn comes, I will need to have published a few decent papers if I want to carry on in my new/old profession.

Publication: it’s been weighing heavily on my mind these past few days. Because I was re-entering research after a four-year career break, it took nearly a year for me to get up to speed. Yes, I orchestrated three very large screens and generated quite a number of solid results in that time, but it took a good dozen months for my brain to re-engage with the knowledge and skills needed to truly hit my stride. And for the subsequent six months, instead of tidying up the unexciting loose ends needed to dispatch my screening paper to the Great Beyond, I’ve been testing the biological promise of a select few key hits that came out. To be sure, I need a bit of biology to elevate the screen to a publishable level, but the characterization has been led more by excitement and instinct than by strategic, manuscript-style thinking. In other words, I have been chasing glimpsed hares through the forest rather than herding my data like sheep.

My boss isn’t helping. He’s been urging me drop everything and tackle those promising leads, the genes that might reveal not just small cogs, but entire machines. And he’s right: this is the fun part, and the activity most likely to lead to the sort of high-profile papers I will need to compete. But personally, I don’t like unfinished business. If something needs to be written up, it really ought to go out the door. Besides a splash of biology, closing the deal will require a lot of hard, unexciting work: analysis, statistics, the tidying up of annotation. Pie-graphs. Lists. Supplementary data. Cajoling a major co-author who’s understandably distracted by a new lab, a new project. Long hours staring at Excel spreadsheets and MetaMorph screen review apps.

Really, I ought to be able to do both: devoting a large fraction of my time to sheep-herding, chasing a few hares where I can. And I keep saying I will –

– after the next fun experiment.

Numerous urban studies have characterized ‘The Broken Window Syndrome’: the notion that abandoned buildings with smashed glass tend to attract more of the same abuse. The phenomenon, it turns out, can be extrapolated to pretty much anything. In his excellent book ‘Blink’, Malcolm Gladwell summarized nicely how defects in one’s environment – graffiti in the streets, for example – can cause people to make a snap judgement about the value of the environment, which in turn attracts crime and further vandalism. Whereas the simple act of cleaning up can lead to a decrease in such negative activity.

Cause for reflection How important is a clean slate?

I’ve often mused about how messiness in the lab can influence one’s experimental processes. It’s definitely true that when my workspace is a disaster area, I find myself cutting corners, rushing a little bit, doing things less carefully than I might otherwise. Similarly, when everything is tidy, I enter into a zen state and become one with my samples.

But this is a pretty rare occurrence. Usually, left to our own devices, the lab gradually degenerates to a chaotic state: styrafoam boxes pile up, all possibly surfaces attract clutter, cardboard freezer boxes accumulate hundreds of rejected, forgotten miniprep tubes numbered one through twelve; the waterbaths secrete algal mats and the incubators, white films of unindentified fungus.

So about twice a year, we have a big lab clean-up. Most people show up at the appointed time (although it seems there is always at least one who rocks up just as the show is over, exclaiming with mock dismay, You’re finished already?). It’s usually a very cheerful affair. This time around we were feeling particularly ruthless; the disposal highlights include:

• several dozen blue boxes full of spent, encrusted miniprep reagents
• a bottle of DEPC-treated water, circa 1987
• about two hundred empty pipette tip boxes (sent for recycling)
• one broken electrophoresis apparatus, complete with fossilized agarose gel inside
• an ancient centrifuge with an adapter for six tubes, whose bench footprint was about one square meter
• four dozen rusty scalpel blades

• three bottles of LB agarose sporting about two inches of iridescent purple and green fur (“Life finds a way.” – Michael Crichton, R.I.P.)

The most exciting moment was when we decided we needed to dispose of all but one of each of the dozens of aged bottles of concentrated hydrogen chloride and sufuric acid huddled around the pH meter.

“Acid to water or water to acid?” I yelled out to all and sundry. (_Acido, acqua; acqua, acido?_ , the Italian contingent muttered amongst themselves to general bafflement). A consensus was eventually reached on the institute rules for such matters, and the toxic bottles were dispatched forthwith. Most satisfactory, as my friend John Cairns) likes to say.

That evening, the elves came to clean and polish the floor. The next morning, the lab gleamed, and everyone walked around in awe. I know from experience that the spell will last for a few days: surfaces will stay tidy, sloshed solutions will be promptly wiped away, tip boxes will be filed on the benchtops at precise right angles. I am positively revelling in my experiments: even my handwriting is neater in my notebook.

But it’s only a matter of time before someone smashes a window.

The heroine of my third novel has a problem with filing: she is incapable of committing to a concrete decision. Instead of compartmentalizing her papers into simple, broad categories, she has arranged her reprint collection into a drawer of hundreds of folders, each labelled with a highly specific topic. A set of information annotated like this will be rich in details, but it might be hard to find what you want quickly, or to get a bird’s-eye perspective of what the overall set contains.

The elusiveness of the binary

Normally when I write fictional characters, I don’t see the similarities with my own personality until I step back. It was only the other day, when I was struggling over annotating one of my validation screens, that I saw what I shared with my heroine.

It should be easy, right? An image of genetically altered cells either depicts something that looks like wild type, or something that differs from it: in other words, a phenotype. When you look at cells, it should be possible to say yes, this is a hit, or no, it is not. But somehow, the mind slides away from the inevitability of the task. I can’t help but worry that any ‘no’ might somehow be eliminating an important clue forever. The first time I went through this smaller subset of fly genes, I found myself ranking things in a painfully analogue fashion – a system of stars from none to four, supplemented with lots of prose marginalia. And even that wasn’t enough: in that seemingly infinite space between no stars and one, I couldn’t help, in a few cases, to write ‘maybe’. Occasionally further undermined by a question mark.

This time through, on the repeat, I was determined to make a clean decision: score each phenotype, compare with the previous run-through’s star ranking, and then decide once and for all: an overall yes or no for each gene.

Well, needless to say I failed miserably. I’ve managed to be ruthless enough to pare it down to only two stars and no maybes, but I couldn’t quite commit to that binary decision. It’s times like these when I start to see the appeal of automated image analysis. In this procedure, all the parameters become numbers, and you introduce a cut-off, under which the answer is no. Even the pain of choosing the threshold is taken from you – it’s all down to statistics, which never equivocate or worry about the consequences of false negatives.

But it’s proving difficult to find any image analysis partners willing to take on my data long-term. I’m already on my third collaborator, but only the other day she broke it off as well – over the phone. It was almost exactly like getting dumped:

“It’s not your dataset – it’s me,” she said. “I’m going in new directions.”

It’s not the size of my dataset that’s scaring them off: it’s the complexity of the textures and structures we want to score. We’ve actually bought a MATLAB license and are thinking about going it alone – but it’s a scary new direction for this squishy biologist.

Tedium: it has its uses.

Biological research is a complex activity requiring many different approaches. We spend a lot of our time making tools – modified plasmids, say, or a transgenic cell line – and the rest of the time testing them in experimentation. The testing is supposed to be the fun part: the creative flexing of our hypotheses, emotionally enriched by the crushing disappointment when they don’t fly and the surges of happiness when they do. The testing, in other words, is the real science.

But spare a thought, if you will, for the tool-making – the necessary foundation for all those dreams. The unglamorous activities that will never, in themselves, lead to fame and glory. Indeed, the repetitive manual labor typically required to make tools is considered to be a lower-caste occupation, often ferreted out to undergraduate interns or outsourced altogether, and scarcely worth more than an acknowledgement in most papers.

I have to admit, though, that there are times in my life when I have not minded taking on such thankless chores personally. When existence in that messy parameter space outside of the laboratory known as ‘real life’ becomes overwhelming for whatever reason, the manipulations of science can be a safe haven. But the catch-22 is that it is precisely when things are particularly turbulent that it difficult to concentrate on higher intellectual pursuits. The mind slides away from rigorous analysis, from creative brainstorming, from the angelic highs of dreaming up the perfect hypothesis and how to test it. So in those cases, mindless tedium can becomes a solace of sorts.

I have a vivid memory of one night in the lab back in Seattle when I was a graduate student. It was about three in the morning, and – following an explosive romantic break-up –I’d found myself almost instinctively getting on my bike and heading for the one place where I knew I could find safety and distraction.

The lab at three a.m. is your kingdom. I recall putting an Offspring album onto the CD player as loud as it would play and working up about a hundred minipreps from pelleted bacteria that I’d been accumulating in the freezer. (I still can’t hear the song ‘Come Out And Play’ without flashing back to that night.) And I found that there was a fearsome joy in the ritual and thoughtless annihilation of so many tubes, cranked through conveyor style: lyse, precipitate, neutralize, extract. Pipette, mix, decant, spin. The DNA crashing out of solution in satisfying white puffs, collecting reassuringly at the bottom of the tube, going clear on drying, then gelatinous in dissolving. Everything controlled and entirely predictable. Quantitate, digest, elecrophorese, photograph. Thoughts somehow both focused and entirely absent from the world in that haze of discrete little steps.

That night, I was a robot – and I needed to be. Equally, in the past week, my cloning work has been the calm in the eye of a little storm, the point of focus that has kept me getting up every morning to incremental nanosuccesses as the new tools slowly progress down that belt. In a few days it will all be over but the sequencing, and my normal experiments will resume.

Perfect timing. At least I hope so.

Over the past few weeks, I’ve stepped in to help a colleague in need. I can’t go into any details, but let’s just say that owing to an astonishingly pedantic visa renewal technicality, a particular country’s immigration policy has necessitated the precipitous (but hopefully temporary) departure of one of our post-docs. He’s due back at the end of July to start a sabbatical position in another lab, on a very tight schedule. And he needs three new DNA plasmids to be ready on his return – buffed, polished and fully verified.

I quite like the thought of being a Cloner For Hire – blow into town with the tumbleweed, step into the bar and, over the sudden silence of the piano’s cessation, drawl slowly, “I hear someone here needs a GFP-tagged point mutant?”

As one, the hushed crowd turns to stare at the post-doc slumped over his whiskey in the corner.

It made sense to ask me. This particular colleague has done a lot on my project and will be a co-author on the paper I’m finishing up now. And he’s offered to stick me on his paper, which is right neighborly. But more importantly, I’m the only one in the lab besides him who really does much molecular cloning – all the geneticists just muck around making their male flies do naughty things with virgins. But I’m old school, and it when it comes to subcloning, I tend to have a green thumb. (You can see where this is going already, can’t you?)

A few hours before hitting Heathrow, the post-doc presented me with a beautiful yellow plastic freezer box containing everything I might possibly need to finish making his three constructs – he’d even crafted little machine-printed labels for the tubes. He was a third of the way through two constructs, though he did mumble something about not everything “quite working” (a comment which would haunt me later). The strategies were pretty complicated – long-range PCR, intermediate subclones – hey, even a three-way ligation! But nothing I hadn’t seen before. I like a challenge. And it was heart-warming – he’d also stocked the yellow box with aliquots of everything I might possibly need on the adventure – ligases, buffers, nucleoside triphosphates, X-Gal, obscenely expensive turbo-polymerases. It was as pleasurable as receiving a picnic hamper from Fortnum and Mason.

Despite this promising start, it all started to go wrong surprisingly quickly. The tube that was supposed to contain half a milligram of gel-purified PCR product was, in fact, entirely devoid of DNA. The vector that was supposed to contain only one EcoRI restriction site appeared to actually have two. Another vector seemed to be contaminated with a smaller plasmid. Undaunted, I made half a liter of LB broth spiked with ampicillin and told my benchmate grimly, “I will finish these clones before the broth runs out. Mark my words.”

When it became clear I’d have to start all over and venture into the post-doc’s personal boxes to find new stocks of plasmids and primers, and into his notebooks to cross-reference them, it was then that it truly hit me how difficult it is to carry on a project after someone else has gone. For in the privacy of one’s boxes, things can get very chaotic very quickly. We all do it: how many of us are guilty of filing away rows of minipreps or ligations that are only numbered, convinced we’ll remember what they are by where they are sitting in the box? It is so easy to cut corners on record-keeping when night is falling, when your timer is alerting you about your other experiment, when you’re late for lab meeting. It’s made me start thinking seriously about barcodes – should I be worried?

Anyway, the post-doc had pretty good handwriting, but he never put dates and it wasn’t always easy to tell exactly what sort of animal was lurking in the tubes – Fragments? Digests? PCR products? Miniprep DNA? I found at least three tubes that seemed to represent the vector I needed, but none of them looked quite right.

“This one is labelled ‘miniprep DNA’, but it’s a liquid at -20,” I told my benchmate, sticking the tube under her nose. “Does it smell like alcohol to you?”

“No,” she said slowly. “Actually, it smells like nectarines.”

“That was from my lunch, silly. How can we work out if it’s alcohol?”

“Color a bit with some dye and see if it floats on water?” she suggested.

“Good idea. Or – hey! Maybe we should try to set it on fire!”

And so forth. The LB/amp broth currently stands at 350 mL, and I’ve just retransformed every starting plasmid into bacteria from scratch, so at least I know what I’m working with.

But the clock is ticking.