Coach Izzo and me

Chalk up another great year for the Michigan State men’s basketball team and coach Tom Izzo. The Spartans were co-champions of the Big10 and won the conference’s grueling tournament. And in the NCAA’s March Madness, they made it all the way to the Final Four, knocking out the top-seeded team in the process.

Being a fan of this team got me thinking: Coach and I have a lot in common. We’ve both been doing our jobs, mostly at MSU, for a long time. Coach Izzo came here as a part-time assistant in 1983, becoming head coach in 1995. I was on the faculty at UC-Irvine starting in 1985, before moving here in 1991.

But the real similarities are deeper and more important:

First and foremost, we’ve both been fortunate to be surrounded by talented and hard-working students who listen to our ideas, experiment with them, develop them in their own ways, and translate them into meaningful outcomes—winning big games and making new discoveries.

That’s not to say there aren’t frustrations along the way: games lost, grants and papers rejected, grinding practice on the court and repetition in the lab, and even occasional conflicts. But our students are usually resilient—they overcome those setbacks and frustrations, and they go on to productive lives as players and coaches, researchers and teachers, and other careers as well.

We also both had mentors who helped us start our own careers. In Coach Izzo’s case, one mentor was Jud Heathcote, the previous head coach who hired him as an assistant. My mentors included my doctoral advisor, Nelson Hairston, and my postdoctoral supervisor, Bruce Levin. Coach Izzo and I also had friends who helped shape our careers early on: Steve Mariucci, who went on to become an NFL coach; and Phil Service, who did important work on life-history evolution.

Coach Izzo and I also both benefitted, I think, from early successes—again, largely due to our students—that helped establish our reputations, allowing us to retain our jobs and thrive by recruiting more talented, hard-working students. For Tom Izzo, it was players like Mateen Cleaves, Charlie Bell, and Mo Peterson who took the Spartans to the Sweet 16 in his 3^rd year as head coach and to the Final Four the next year, and who won the 1999-2000 National Championship. For me, the early students included Judy Bouma, Felisa Smith, John Mittler, Mike Travisano, Paul Turner, and Farida Vasi, and postdocs Toai Nguyen and Valeria Souza.

Coach Izzo has also had assistant coaches and staff, who I imagine do a lot of the heavy lifting. While some might eventually become head coaches of their own teams, many others labor in relative obscurity. In a similar vein, I’ve had outstanding lab managers including Sue Simpson, Lynette Ekunwe, and—for over 20 years, before retiring last year—Neerja Hajela.

Coach Izzo and I have both had deep benches—students who helped the team succeed without being in the limelight themselves. For Coach Izzo, they include the walk-ons and others who see limited action in games, but who compete against the starters every day in practice, helping everyone become even better. I think of three undergraduates who joined my lab when it was just getting started in Irvine (all Vietnamese refugees, by the way) who asked if they could work in my lab. Trinh Nguyen, Quang Phan, and Loan Duong prepared media and performed experiments like some incredible three-brained, six-handed machine, setting a high standard for everyone who followed in their footsteps.

Coach Izzo and I are nearly the same age. Retirement might be easier, but neither of us is ready for that. It’s too much fun when you’ve got talent to encourage and guide like Cassius Winston, Joshua Langford, Nick Ward, Xavier Tillman, and Aaron Henry—and on my team Jay Bundy, Kyle Card, Nkrumah Grant, Minako Izutsu, and Devin Lake.

Of course, there’s more that Coach Izzo and I have in common—we were lucky to be born into circumstances that allowed us to pursue our dreams without the obstacles that many others face.

Last but not least, Coach Izzo and I have had supportive partners who’ve accepted our peculiar obsessions and the long hours and frequent travel that our work entails.

Go Green! Go Students!!

Thank you, Neerja

Neerja Hajela has worked with me for over 22 years as a technician and lab manager. She is extremely skillful, diligent, organized, and dedicated in her work. On top of all that, she is a wonderfully kind and warm person. Now, this is her last week before she retires.

It’s impossible to put into words all that Neerja has done for me, for everyone in the lab, for the LTEE, and for my collaborators. But let me mention a few of the things she has done.

By keeping the lab running in a smooth and orderly fashion, Neerja has enabled me to spend more of my time thinking about science, writing papers, giving talks, etc., etc. We scientists sometimes complain that we have too much to do, and so we joke that we want to clone ourselves. Well, I’ve done better—I’ve had Neerja.

Those of us with labs know that our institutions take laboratory safety very seriously, as well they should. Neerja runs such a tight ship that, on many occasions after inspecting our lab, the safety officers have made comments to the effect that they wish all labs were as neat, clean, organized, and safety-conscious as ours.

One of the challenges of the long-term evolution experiment (LTEE) with E. coli is freezer management. We now have over 30 years of samples, spread over half a dozen freezers, which provide a record of past evolution. Neerja has overseen this ever-growing collection with extraordinary care and dedication. The samples provide critical backups that allow us to restart the LTEE from a recent milestone when mishaps occur, and they provide unique research materials such as when new technologies emerge. A case in point: Michael Desai wrote me a few years ago with a request. In essence, he wanted all of the LTEE samples for metagenomic sequencing. All of them—from each population and every generation with saved samples. Since I started the LTEE in 1988, we’ve always saved duplicate samples, with one of them being a backup to be opened only in an emergency. I could send Michael the backups, perhaps, but that didn’t seem like a good idea. So I decided we should make additional sets by going into the ~1500 key samples spread over several freezers; taking a subsample of each and culturing it to produce a larger sample; splitting the new culture into ten sub-cultures; and freezing those to provide a new set for Michael as well as other sets for collaborators and institutions. It took Neerja many months to accomplish all of this, but as always, she did it with great skill and care. (Oh, and you can read about the results of Michael’s request here.)

Last, but surely not least, Neerja has done more of the daily transfers of the LTEE than anyone else. She performed her first LTEE transfer on February 5, 1996, and since then she has done well over 4,200 daily transfers. (Thanks to Zachary Blount, who went through the LTEE lab notebooks for its 30^th birthday.) And when Neerja hasn’t done the transfers herself, she has organized who else is responsible for each and every day’s transfers.

Thank you, Neerja, for all that you have done for me, for everyone in the lab, for the LTEE, and for science. Everyone in the lab joins me in wishing you and Ravindra all the very best in your retirement and new home!

 

[Neerja Hajela]

[Here’s Neerja doing yesterday’s LTEE transfer]

[Neerja pointing at two of her LTEE entries: her first transfer on February 5, 1996, and the one from yesterday July 30, 2018. The lab notebooks in the background record the daily transfers since she joined the lab.]

[Close-up of Neerja’s first entry.]

[Neerja’s protocols for the LTEE, mounted in the lab, so nobody makes a mistake]

[And in case that doesn’t work, here’s Neerja enforcing lab discipline]

[The LTEE transfer board from earlier this year]

[Horsing around Tanush’s plate tower]

[Neerja making copies of freezer samples]

[Neerja and me]

Some Wrinkles in Time

Today is another milestone for the E. coli long-term evolution experiment—the LTEE, for short. I did the 10,000^th daily transfer today at about noon.

[Yours truly, doing the 10,000^th LTEE transfers. Technician Neerja Hajela is keeping a close eye on me, and with good reason. Photo by Thomas LaBar.]

Some of you will remember we just celebrated the LTEE’s 29th birthday a few weeks ago, on February 24^th. And if you’re quick with math, you might be thinking: “Wait a second: 29 years times 365 days per year is a lot more than 10,000 days. Have Lenski and his team screwed up?”

The answer is both yes and no. Let me explain.

The LTEE began on February 24, 1988 [1, 2].

From February 24, 1988, to March 13, 2017, equals 10,609 days on which we could have done transfers. But we’ve only had 10,000 transfers. What happened to those other days?

In short, the bacteria spent the 609 “lost” days in a freezer at –80°C or in a refrigerator at 4°C.

One chunk of days was lost when the LTEE was moved from my lab at UC-Irvine, where I started the experiment, to MSU, where it is today. Moving a lab is difficult: it requires moving people, moving equipment and materials, often renovating space, obtaining new supplies and equipment, hiring new people, and trouble-shooting and otherwise getting everything organized to resume work [3].

We lost 191 days from April 8, 1992, when the 10,000-generation samples went into the freezer at UCI, to October 16, 1992, when the LTEE restarted from the frozen samples at MSU.

Most of the other days have been lost as a result of various accidents. I’m often asked, when I give talks on the LTEE, how we’ve kept the experiment going so long without contamination, broken flasks, equipment failure, etc.

The short answer is that we haven’t. Many accidents have happened along the way.

There are 3 main types of accidents, each of which involves a different sort of interruption and recovery.

Little mistakes: Sometimes a flask has a hairline crack; when you take it out of the incubator the next day, there’s just a puddle of salt on the bottom. Or maybe someone knocked over a flask while doing the daily transfers. In cases like these where a mistake occurs that is immediately recognized, we go back in time (and lose) one day.

How do we do that? Each day, after the transfers have been made, we don’t immediately discard the previous day’s cultures. Instead, we put them in a refrigerator, where we can use them to restart the experiment after these little mistakes. The bacteria have finished growing long before each day’s transfer, so they are in stationary phase, and their metabolic activity is even lower sitting there at 4°C. Restarting the populations from the refrigerated cultures is a perturbation, of course, but a tiny one in the scheme of things.

When these little mistakes happen to one population, we go back a day for all the populations. We do that so that the rhythm of the experiment, which involves quality-control checks and freezing samples at regular intervals, is the same for all of the populations.

Bigger slipups: Another sort of problem can occur if the entire experiment is compromised in a way that is not immediately recognized. For example, the autoclave might not be working properly, and we realize that bottles of media that we’ve been using for a few days are contaminated. In that case, the cultures stored in the refrigerator won’t help us.

But we don’t have to start the LTEE all over at t = 0. (If we did, then the experiment wouldn’t be here today!) Instead, we go back to the last time that we froze samples, just like we did when we restarted the experiment after the move from UCI to MSU. Importantly, we restart the LTEE from whole-population samples, not individual clones, so that we do not lose the diversity that is present in an evolving population.

Of course, moving the bacteria into and out of the freezer is a perturbation, involving the addition of a cryoprotectant, freezing the cells, thawing them, and re-acclimating them to the conditions of the LTEE. Still, it happens only occasionally. Moreover, all of the samples used in competitions or other assays go into the freezer, come out, and are re-acclimated to the relevant conditions before measurements are made.

Dreaded cross-contamination: The third kind of accident is when bacteria from one LTEE population “migrate” into another population. That’s not supposed to happen, because it compromises the statistical independence of the populations, which are units of replication on which many analyses rest. I worried about this issue before I started the LTEE, because one of the central questions that motivated the experiment is the reproducibility of evolution. And I’m glad I worried about it. Fortunately, there was a pretty easy way of dealing with this concern from the outset.

Six of the 12 populations started from cells of an ancestral strain, REL606, that cannot grow on the sugar arabinose; they are phenotypically Ara^–. The others started from cells of a mutant, REL607, that can grow on arabinose; these populations are Ara⁺. There is no arabinose in the LTEE environment, and the mutation that allows growth on arabinose has no measurable affect on fitness in that environment. However, when Ara^– and Ara⁺ cells grow on Tetrazolium Arabinose (TA) agar in a petri dish, they make red and white (or pink) colonies, respectively.

[Mix of Ara^– and Ara⁺ colonies on TA agar.]

The arabinose phenotype serves two important purposes in the LTEE. First, we use it to estimate the abundance of competitors in the assays we perform to measure relative fitness. To that end, we typically compete an evolved Ara^– population sample against the Ara⁺ ancestor, and vice versa. Second, with respect to the possibility of cross-contamination, we alternate Ara^– and Ara⁺ populations during the daily transfers. The idea is that, if an accidental cross-contamination does occur, it will likely involve adjacent populations and lead to cells that have the wrong phenotype (i.e., produce the wrong-colored cells on TA agar) in a population. So we check each population for that phenotype whenever we freeze samples.

When we find one or more cells that produce the wrong-colored colony, we have to figure out what to do. There are various additional checks that we can perform, especially nowadays when DNA sequencing has allowed us to discover many mutations—additional markers—that uniquely identify each population. In particular, these extra markers have, in recent years, let us distinguish between “false alarms” (new mutations that affect colony color on the TA agar) and actual cross-contamination events. In any case, when we’ve had suspected or confirmed cross-contamination events, we restart the invaded population from the previous sample [4]. We then typically monitor that population by plating samples periodically on TA agar, to make sure it didn’t have a low frequency of cross-contaminating invaders even before that earlier sample was frozen. As a consequence of restarting invaded populations, some of the LTEE populations are 500 generations (or multiples thereof) behind the leading edge.

So today’s 10,000^th daily transfer applies to some, but not all, of the LTEE populations.

Despite these precautions and procedures, I worried that somehow we had slipped up and there were undetected cross-contamination events. Maybe there had been an especially fun party one Friday night … and on Saturday someone forgot the protocol and transferred all six red Ara^– populations in a row before moving on to the six white Ara⁺ populations. In that case, a cross-contamination might occur but not be detected. So I was thrilled when we sequenced hundreds of genomes from different generations of the LTEE populations and there was no evidence of any cross-contamination. Have I mentioned all the terrific people who have worked with me?

One of the unsung heroes of the LTEE is my technician and lab manager, Neerja Hajela. She has worked with me for over 20 years now, and she’s probably done more daily transfers than everyone else combined.

[Neerja Hajela, technician and lab manager extraordinaire.]

By the way, there were not 12, but 15, flasks in the trays while I was doing the transfers. What’s going on with that?

[The 15 LTEE flasks in the incubator.]

One of the extras is a blank—a culture without bacteria. If the medium in that flask is turbid the next day, then “Houston, we have a problem.” Another of the extras is a population we’re calling Ara–7. It was spun off population Ara–3 after we discovered—many thousands of generations later—that one lineage in that population had gone extinct for some reason that we do not understand. You can read more about that here. Ara–7 doesn’t count as one of the “real” LTEE populations, but it might prove useful in comparison with Ara–3 at some point in the future.

And the third extra? Remember what I said about cross-contamination? Well, we recently discovered a cross-contamination event in which cells that made red colonies on TA agar were found among the white-colony-forming cells of the Ara+1 population. Postdoc Zachary Blount confirmed they weren’t new mutants that made the wrong-colored colonies in Ara+1; instead, those cells had specific mutations that showed they came from population Ara–1, meaning they were cross-contaminating invaders.

[Zachary Blount, aka Dr. Citrate.]

So we restarted Ara+1 from its previous frozen sample, monitored it by plating cells on TA agar, and … alas, up came some more of those red invaders. It’s interesting, in a way, because Ara–1 is one of the most fit LTEE populations, while Ara+1 is the very least fit, which means Ara+1 is especially susceptible to invasion from its Ara–1 neighbor in the daily transfers. Anyhow, we then restarted Ara+1 going back in time 1000 and 1500 generations—hence, the extra flask—and we will monitor those for a while by plating samples on TA agar. If neither of them shows any sign of invaders for several weeks, then we will continue only the one with the fewer “lost” generations and drop the other.

There’s one other little issue related to keeping time in the LTEE. Every day, we remove 0.1 mL from each flask culture and transfer it to 9.9 mL of fresh medium. That 100-fold dilution allows the bacterial population to grow 100-fold before it depletes the available resources. And that 100-fold growth corresponds to log₂ 100 ≈ 6.64 generations. But we round it up a tad to 6.67 generations, so that every 15 transfers equals 100 generations [5].

In any case, our fielding percentage (baseball jargon for the ratio of plays without errors to total chances on defense) is 10,000 / 10,609 ≈ 0.943. If we exclude the lost days associated with the move from UCI to MSU, then the percentage rises to 0.960. Not bad, not bad at all. Did I mention the terrific people who have worked, and are working, on the LTEE?

This post’s title is a play on the novel A Wrinkle in Time by Madeleine L’Engle.

[1] I first started the LTEE on February 15, 1988, but I then restarted it on February 24, because I got worried that the first arabinose-utilization mutation I had selected, which serves as a neutral marker, wasn’t quite neutral.

[2] So the LTEE experienced a leap day in its very first week!

[3] I was fortunate that three experienced graduate students—Mike Travisano, Paul Turner, and Farida Vasi—moved to MSU even before I did to help set up the lab, and that our research was allowed to continue in my UCI lab—led by technician Sue Simpson and John Mittler, who was finishing his PhD—after I moved in late December, 1991.

[4] To keep all the populations in sync with respect to the freezing cycle, we restart the others at the same time, too. Of course, for the others, we don’t go back in time—we use the latest sample, where the cross-contaminated population was discovered during the quality-control checks associated with the freezing cycle.

[5] In fact, 6.67 generations per day might be a slight underestimate given the possibility of turnover during stationary phase. Moreover, every lineage with a beneficial mutation that sweeps to fixation goes through more than the average number of generations, since each mutant lineage starts as one cell among millions.

Happy 27th birthday to the LTEE!

The title says it all:  today is the 27^th birthday of the long-term evolution experiment (LTEE) with E. coli.

Well, the title doesn’t really say everything. I also want to give thanks to the many people—not to mention the trillions of bacteria—who have made it possible for the LTEE to keep on going and giving.

So thank you to all of the students, postdocs, and colleagues with whom I’ve collaborated on this project. There are too many to list here, but you will find their names on the papers that have come from the LTEE. I’ll call out just two, on this occasion, for special thanks. Dom Schneider has been an amazingly talented and generous collaborator for so many years—in fact, our first co-authored paper on the LTEE dates back to 1999. And Neerja Hajela has worked with me for 20 years now, and she is the most organized, dedicated, and all-around wonderful technician and lab manager that one could ever have.

Special thanks, too, to Madeleine Lenski, who has tolerated my long-term affair with the LTEE, and who wisely advised me to keep it going on one or two occasions when I was looking in other directions.

[The image below shows the abstract from the first paper on the LTEE, which appeared in The American Naturalist in 1991. It is reproduced here under the doctrine of fair use.  Some of the conclusions have changed a bit as the LTEE has had more time and we’ve gathered more data—that’s science!]