On handling Chlamydomonas

As part of my doctoral research, I'm working with the unicellular algae Chlamydomonas reinhardtii as one model genetic species in a predator-prey system (the predator being the model ecological species, Daphnia pulex).  Recently,  Kyle Hernandez, a post-doc in the department who is interested in the genetics of function-valued traits, has started working with me.  One of our goals is to generate a large QTL mapping population starting with wild-type strains that differ in some respect, in this case, average cell size through the day.  Algae cell size important for two major reasons: first because small-bodied Daphnia selectively consume the algae based on cell size, but large Daphnia do not; and second because cell size--hence surface-area to volume ratios--is central to various physiological processes, such as nutrient uptake rates.

Generating a mapping population requires crossing the parental strains, separating out individual zygospores, then individually separating the four daughter cells produced when the zygospores mature, to start each line in the mapping population.  While there is an excellent Journal of Visualized Experiments article on making Chlamydomonas crosses, Kyle and I have run across many questions during our trials of generating crosses:

1. Is plating the cells out on COMBO media with 10% normal nitrate (i.e., very low concentration) sufficient to trigger sexual reproduction.
• Yes, it is
2. After the one-week period of incubation in the dark, will we actually be able to tell the zygospores from vegetative cells?
• Yes, at least with the lines we are crossing, the zygospores are larger and a glowing golden color distinct from the smaller, green vegetative cells...super obvious
3. Are you kidding me?  How the world do people handle individual Chlamydomonas cells???
• No, seriously, is this actually possible?
• Yes.

So that last point has been our big learning experience over the past couple of days.  The process requires practice moving very slowly and carefully, but it also requires making the glass needles correctly.  Specifically, the first needles we pulled were far too short and far too inflexible to be of any real use: the slightest twitch and the needle tip will plunge right through the agar (taking with it any cells with which you might be trying to work).  The key is to pull glass needles that look something like this:

Yes, it is difficult to see the needle, because it's ~20-30 microns across out towards the hooked end (the hook is nice for pulling cells along).  The more important point is noting the length of the thin portion: in this example it's over 2cm long.  The long, very thin portion allows a lot of flex to the needle, to the point that the surface tension of fluid on the surface of the agar is enough to hold the tip down and deliberate pulling up is required to overcome the spring to lift the hook.  Once I got the appropriate needle length pulled, it became much easier to cut a little bunch of zygospores from the incubation plate; place them on the maturation plate; then slide the cluster around repeatedly until individual cells popped off and could be slid into place.  

Kyle and I could not find this information anywhere on the Internets, and so hope that this information is helpful to anyone starting to work with Chlamy (or other single-cell species that require such techniques and tools)!

Funny

A student missed class this morning, then sent an email to the other students trying to figure out how to weasel out of a commitment.  The email was also sent, unbeknownst to the absent student, to the faculty sponsor for the class.  The faculty sponsor forwarded the email to the instructor, the other TA, and me, then replied to absent student to point out the clause of the syllabus that applied.  I later replied to absent student saying that the commitment stood; absent student replied that, "Professors at UT have a troubling habit of sharing email correspondence with people to whom it was not addressed..." 

So, naturally, I forwarded his reply to the faculty sponsor!  Fun!

Data consolidation

About nine months ago I realized that I had too many data/idea sources floating around, a data fragmentation problem of my own: a wet-lab notebook for molecular work, a live-lab notebook for work with critters in the lab, a couple of clipboards with scrap paper and doodles, a couple of doodle notebooks for different topics, plus a field book.  I consolidated everything down to a single lab book and a field book...plus clipboards...plus the chalkboard...plus gel images on a non-connected desktop in the wet-lab...plus...AUUGGGGHHH.  Things were better for a bit, but still fragmentation took over.  (Plus I couldn't search everything...but that's a side-story.)

This past week I began trying to solve the information fragmentation electronically.  In part driven by by the release of Google Drive, I started with Google Docs (and the Drive once available).  With the Google services I could carry my laptop everywhere indoors (the fieldbook is the only thing to remain long-hand), and just type lab notes, ideas, etc. and access them from anywhere with an internet connection.  While there were several things that I liked about the setup, there was more that I didn't like.  Most importantly, if you create a file in Google Drive (formerly Docs), there's no program outside of Drive with which you can edit the file.  Yes, you can download as a format that is editable on a local machine, but then if you want to edit the revised file in GDrive, you have to convert it back!  Data fragmentation was the driving problem, and adding Google's format(s) to the mix only makes fragmentation worse.

So what's a fella to do?  My current, and hopefully final, solution is to continue focusing on a single system, in this case, Microsoft.  Yes, the iPad will continue to play a role for reading and lightweight data use, but after working with OSX and various Linux flavors extensively over the past couple of years, I have returned happily to Windows for the vast majority of my computing requirements.  Shit just works; there's no fighting, no trying to figure out a suitable work-around, and so-forth.  In the old days there were stability issues, but it's been so long since I've crashed a Windows machine that I can't even remember when it last happened.

And so I have everything synced with SkyDrive, which also has online versions of Word through OneNote available.  I can keep wet, live, and computational lab notebooks in OneNote, access everything in standard Word, Excel, or Powerpoint formats from any connected computer.  Files are actually synced locally, even if created online, which is not possible with GDrive.  The next phase is to integrate my new Windows Phone (a Lumia 900, purchased earlier today) so that I can snap photos in the lab and directly drop them into notes.

I really hope it works!

Missed a day...

...for reasons passing understanding.  The lack of a posting yesterday was rooted in constant programming problems (damn you MemoryError when building gigabyte-sized Python dictionaries!).  Hopefully that is being solved as I type this...

Chlamydomonas mating

One of my primary study organisms is the model algae, Chlamydomonas reinhardtii.  I am currently working with a postdoc from the Juenger lab on a project creating a large QTL mapping population starting from two wild-type parental lines that differ (by ~5-fold) in size. The goal (in my mind) is to reach the F12 generation so that the genome is well-recombined and should permit high resolution mapping of loci related to interesting traits (e.g., fitness under different nutrient conditions; or susceptibility to predators such as my other study organism, Daphnia; both traits are regulated by cell size).  At any rate, today was the first day of actual crosses...it looks like it may have gone well, given the jump in cell motility after suspension and obvious mating after mixing the lines.  But we shall see!

Trying for consistency

Last week, Atrios celebrated ten years of bloggering; during that decade, except for a couple of short vacation periods, I think he posted at least once every day.  Impressive.  During that same timeframe, I think I've had ten or so various blogs that always get pushed aside when things (e.g., field season, lab season, etc.) really start humming along.

This blog is yet another attempt at consistent blogging: I am going to attempt to write at least one post every day for as long as possible.  Who knows...maybe I'll forget tomorrow, or maybe I'll make it a while.  We'll see!