Biocurious

/ a biophysics blog

SciBarCamp report: Digital lab notebooks

Posted 16 March 2008 by PhilipJ under &

The first SciBarCamp is going on this weekend at Hart House at the University of Toronto. The basic idea of SciBarCamp is that of “a gathering of scientists, artists, and technologists for a weekend of talks and discussions.” The kinds of things that have been discussed this weekend are the forefront of science (quantum gravity, synthetic biology, open access, scientific software), and the interactions with science and technology and art. There’s an extremely interesting mix of people here today, from quantum information theorists from the Perimeter Institute to social scientists from OCAD, grad students, science writers, musicians, etc. Quite the eclectic group.

The “un“conference opened up with what was perhaps the most interesting discussion for me. It was by Corie Lok from Nature Networks and John Dupuis from York University on “Science 2.0”, or basically how technology is changing first the way we do science, and then how we publish and track science. Things like Connotea and del.icio.us were discussed as ways to keep track of relevant journal articles, but the discussion was dominated by discussion about keeping track of your experiments, and how this is changing in laboratory environments.

Many labs are moving to an all-digital lab notebook. Gone are pens and paper as the primary means of keeping track of your experiments, and they are instead being replaced by digital equivalents, such as wikis or some other, commercial software (which I unfortunately didn’t catch any names, but a google search brings up all kinds of examples). The advantages of such a “notebook” are clear: entirely searchable, easily referenced with hyperlinks, the ability to keep digital images and snippets of code, etc. There is also an advantage which hadn’t been discussed, but is something I’ve long thought about: legibility! If you’ve ever looked in another scientists notebook, you will quickly find that their writing may be atrocious (guilty as charged), and it quickly becomes an exercise in frustrating trying to interpret what was written.

Perhaps not surprisingly, this is being embraced in a big way by the big pharma companies, where it is crucial to have detailed records, and to keep them for a long time (something like the lifetime of a drug being sold + ten years). But there’s an advantage to graduate students in academic laboratories that hadn’t been addressed in the discussion, that came to me as I was talking about this to another grad student friend. It’s that of the copies. I graduated from SFU with my Master’s less than a year ago, but I could tell you basically no specific details on some of the more mundane, day to day experiments that I carried out. I’m sure the same thing happens to those who graduate with a PhD, and leave their lab notebooks behind. I’ve decided I want to keep this kind of information, and I’m still early enough in my PhD that it won’t be too hard to change.

To the other scientists reading this blog: have you changed to a digital lab notebook? Have you tried the switch and failed, or have things gone smoothly? What software did you decide on? I’m leaning towards a wiki, and I’ve decided it will be necessary to keep a paper notebook as well, but to make it degenerate information which will, at the end of every day, always end up in the digital notebook as well.

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Carbon makes how many bonds?

Posted 9 September 2007 by PhilipJ under &

Physicists have a knack for thinking we know everything, or at least that everything else is easy enough to learn, since, well, everything else isn’t physics. I’ve had the sense to not give in to this delusion too often, even though I decided that I could hack it as a PhD student in a chemistry department despite not knowing any chemistry. In my defense, my PhD program is called Chemical Physics, so this shouldn’t be entirely out of my realm.

Prior to accepting the offer here at the University of Toronto, it was agreed that my background would preclude me from TAing a number of courses, particularly those with the words “organic” and “inorganic” in them. Not that I couldn’t learn these subjects, the physicist in me likes to tell myself, simply that they are different enough from my formal training that I can’t pick them up without a reasonable amount of time and effort.

TAships were handed out a couple of weeks ago. As it happens, I was given the computational laboratory section of Intro Organic Chemistry I. An organic chemist friend of mine cheekily asked, “So, how many bonds does Carbon form?” Yes, I do know the answer. No, I am definitely not who you want teaching students about organic chemistry.

Luckily for everyone (though mostly the undergrads in Intro Organic Chemistry I!), I’ve since been switched to a physical chemistry course. The first disaster of PhD-life averted.

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Piled higher. and Deeper.

Posted 12 May 2007 by PhilipJ under &

My Master’s thesis is being bound somewhere (or so I’m told), and my days left at Simon Fraser University are winding down. For the next month I’ll continue on the optical tweezers I helped build, extending various short biomolecules to figure out their elastic properties. Leading up to more exciting work, I’m trying to reliably tether and pull on extremely short pieces of double stranded DNA, which is challenging from a technically standpoint. I’m not sure how much it helps, but I’ve started using smaller beads (1 μm), but even then it is very difficult to reliably tether and measure single molecules between the beads. Too many DNA molecules makes the beads stick hopelessly tightly together, and too few means I’m sitting around all day waiting for the unlikely tethering event to occur. But in either case, it’s nowhere near as unpleasant as shitting yourself for science.

But I am looking forward to a change, scientifically. Single-molecule biophysics has only in the past few years become a mature area that is answering really interesting and novel questions, but, for whatever reasons, it isn’t for me.

I hadn’t mentioned at the time, but my trip to Toronto was really two-fold: the Chemical Biophysics Symposium was a great small conference, but I was also there to visit labs as a prospective Ph.D. student — in the chemistry department!*

All of the labs I visited were doing interesting things, but you can only choose one. Instead of looking at the force-mediated dynamics of biomolecules as I have been for the past couple of years, I’m switching gears to look at (and maybe control) the dynamics of proteins on ultrafast timescales. The Miller lab is an ideal place to do this, where they’ve recently been optically controlling isomerisation of retinal in the protein bacteriorhodopsin with intensity- and phase-shaped light. From a recent paper (subscription required to read the full article):

By modulating the phases and amplitudes of the spectral components in the photoexcitation pulse, we showed that the absolute quantity of 13-cis retinal formed upon excitation can be enhanced or suppressed by ±20% of the yield observed using a short transform-limited pulse having the same actinic energy. The shaped pulses were shown to be phase-sensitive at intensities too low to access different higher electronic states, and so these pulses apparently steer the isomerization through constructive and destructive interference effects, a mechanism supported by observed signatures of vibrational coherence. These results show that the wave properties of matter can be observed and even manipulated in a system as large and complex as a protein.

The details of my project haven’t been worked out, but it is bound to be extremely cool.

* It’s a good thing we recently changed the tagline from “physicists exploring an interest in biology”!

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You can call me...

Posted 26 April 2007 by PhilipJ under &

Master.

I’ve been busy. Really busy. Writing, editing, rewriting, fixing figures, fixing figures again, and today, defending. That which is my Master’s thesis has been given the okay, though there are a few requisite corrections to finish off over the next few days. The defense went well, except when trying to recall the functional form of the Reynolds number (it wasn’t pretty).

Now that I’m almost in the clear, it should mean I’ll get back to much more regular blogging. I think I’ll start, partly as a way to teach myself the relevant background information, on the science behind the new direction I’m taking for my PhD.

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Ten simple rules for selecting a [lab]

Posted 3 December 2006 by PhilipJ under &

PLoS Computational Biology seems like lists as much as I do. This time around in Ten Simple Rules for Selecting a Postdoctoral Position they’re offering advice to those soon to be finishing their PhDs. In fact, the majority of the list is good reading for those who are about to start a graduate degree as well, so click here to read the whole list, no subscription required.

Since I’m in the proccess of figuring out labs in which I’d like to apply for my own PhD, I’ve spent a lot of time thinking about the combinations of rules 5 (Choose a Project with Tangible Outcomes That Match Your Career Goals) and 10 (Learn to Recognize Opportunities). Taking on exciting new projects by definition means you are charting in unknown waters, and tangible outcomes are no longer guaranteed (and even for a lot of me-too science). The editorial notes,

[f]or a future in academia, the most tangible outcomes are publications, followed by more publications.

How does one successfully ballance tangible outcomes (publishing papers [that people will read]) with choosing a lab doing novel and interesting science, and what are some safeguards from overly-ambitious “opportunities” that take too long to produce those tangible results? I’ve seen a number of really excellent talks (most recently at the Frontiers in Biophysics retreat), only to realise by the end that the number of dead ends graduate students (over a few generations) followed on these projects numbered much larger than the successes.

I’ve started learning the lessons, but I don’t yet have the answers.

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The perils of research

Posted 20 October 2006 by PhilipJ under &

For the past year, I’ve been spending the majority of my time starting a collaborative project between my lab and a group of microbiologists at the school across town. This past week, unable to really get things off the ground, I threw in the towel.

In past research-related posts, I was rather coy about what I was doing, and when I went to Japan, I didn’t fill everyone in on the project deemed interesting enough to fly me across the pacific. Unfortunately, basically since then, I’ve made no significant progress. To anyone wondering what I’ve been doing, and what I’ve started doing instead, here goes.

If you want to get DNA inside of cells (for, say, growing new proteins, or making a strain of cells that is resistant to certain antibiotics), there are only a couple of options. For E. coli, one commonly incubates cells with your DNA of interest, and either zap them with electricity, or heat them up quite rapidly in warm water. Both have the effect of making the cell membrane significantly more permeable to, well, everything, and DNA somehow passes through and is then able to get expressed by the cell’s transcription and translation machinery. As you can imagine, the cells don’t like this, and many die. Those that don’t, however, now have new DNA inside of them.

There are other cells, however, which have special complexes which span the cell membrane that can actively bind and internalise DNA, for reasons which are still contested. Two of the main ideas are food and gene transfer. As a nutrient source, DNA can be broken down into individual nucleotides, which could then be used for any number of dogmatic processes. As a gene transfer mechanism, uptaken DNA could be incorporated into the genome, or simply expressed as is, as a way to gain new genes. The common bacteria Bacillus subtilis is one example of a bug which is naturally able to internalise DNA.

In the bacterium Haemophilus influenzae, the story is even more interesting. Haemophilus is a gram-negative (having two membranes) bacteria, so any DNA uptake machinery is going to have to span two separate membranes (B. subtilis’ need only span one). Complicating things even further, to get efficient DNA uptake, a 9 basepair Uptake Signaling Sequence (pPIU should now make sense to you!) is required, which is thought to be perhaps a binding site on a receptor protein on the cell surface. It isn’t entirely clear, but without this sequence, uptake basically doesn’t occur at all.

Given our laboratory’s ability to tug on single DNA molecules, a group across town got excited and contacted us about starting the collaboration to look at DNA uptake with live cells at the single molecule level. It didn’t sound hard to begin with; make some DNA that has both a USS and will be suitable for uptake measurements in the optical trap, and find some way to immobilise cells to a surface. To anyone who has never started a brand new project before, please take note: everything is hard to begin with.

Though I have no formal training as a molecular or microbiologist, I didn’t see any reason why I couldn’t do this kind of work, even if there might be a learning curve to start out. Surprisingly, the DNA cloning steps took only a month or two, but immobilising the H. influenzae cells to a surface has been much trickier than we hoped.

We wanted to stick cells to the surface of beads, either by non-specific interactions, or via antibodies to surface proteins found on the cells, as cells don’t usually live long in the focus of the optical trap, and our micropipette tips are already finicky enough without clogging them with cells. By immobilising the cells onto beads, we could save ourselves a lot of hassle. Our lab has all kinds of beads with different coatings (carboxy groups, protein G, anti-digoxigenin, streptavidin), but the cells didn’t seem to bind naturally to any of them. They also don’t seem to immobilise on glass coverslips.

Up next was contacting another lab for antibodies to common outer membrane proteins. I was sent three different kinds of sera, made to target the unimaginatively named P2, P4, and P5, all residing on the outer membrane, with P2 accounting for as much as 20% of all membrane proteins! This sounded promising.

Through a number of tests (dot blots), I can coat beads with these antibodies, and the cells seem to bind the antibodies as well, but at no point have I seen cells bind to antibody-coated beads. Never. Zero times! I’m not able to explain why.

Finally, we decided I should attempt grabbing cells in the pipette tip to test for uptake events; and should I see any with reasonable frequency, to brainstorm new ways to attempt to immobilise our bacteria. After testing a number of cells (and clogging a few pipette tips along the way) without observing any kind of uptake events, it was decided to give things a break.

I’m not going to lie, I’ve had a really hard time trying to stay positive these past couple of months as experiments continued to fail. It is no secret that science is hard, or that some (most?) experiments end in failure or with entirely ambiguous results, but it is so easy to forget that when they aren’t your own experiments.

So, what now? I’m in the physics department, so they unfortunately won’t really care that I’ve put a lot of work into something that we’re more or less putting on the back burner for a while, since that something happened to be primarily biology. I’m also a little too proud to write a masters thesis without feeling like I’ve accomplished something worthwhile, so I’ve changed to a new project involving single-stranded DNA which can form neat secondary structures, looking at the unfolding of the structure under different conditions. A bit less flashy, and taking so long for a masters degree doesn’t feel great, but with a little luck things will work out.

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