Category Archives: Science

Some Wrinkles in Time

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

REL doing LTEE transfer 10,000 with Neerja keeping a close eye on me

[Yours truly, doing the 10,000th 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 24th. 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 Araand Ara+ cells grow on Tetrazolium Arabinose (TA) agar in a petri dish, they make red and white (or pink) colonies, respectively.

Ecoli-plate

[Mix of Araand 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 10,000th 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 13-Mar-2017

[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?

Flasks LTEE day 10,000

[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 13-Mar-2017

[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.

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Who Knows Where the Time Goes

Today is the 29th birthday of the long-term evolution experiment (LTEE). As I wrote on Twitter: “May the cells live long & prosper, both in & out of the -80C freezers.” I hope they—and the rest of the world—will be evolving and improving long after I’m gone.

Anyhow, after my tweet, Luis Zaman asked for a picture of me on my own 29th birthday. (I started the LTEE when I was 31.) Alas, I don’t have one. But I’ve found some pictures from around that time—including just before and after I moved to UC-Irvine to start my first faculty position, and over the next few years up to about the time I started the LTEE.

Summer, 1985: This photo is from Amherst, Massachusetts, where I did my postdoc with the amazing Bruce Levin, who hosted a goodbye party for us. From left to right: Ralph Evans, a brilliant graduate student and dear friend, who died tragically just a few years later of brain cancer. My beautiful wife, Madeleine. Our one-year-old daughter Shoshannah, being held by forever-young Bruce. Yours truly, holding our three-year-old son Daniel. And Miriam Levin, an art historian.

amherst-goodbye-party-summer-1985

October, 1985: Shoshannah on my shoulders at the San Diego Zoo, a few months after we moved to Irvine.

october-1985-san-diego-zoo-with-shosh

March, 1986: First-year faculty member burning the midnight oil in our Las Lomas apartment at UCI. Working on a paper? Or getting ready to teach 700 students the next day? (Two sections of Ecology, a required course for Bio Sci majors, with an hour to recuperate in between. It was well worth it, though, because one of the students in one of the many quarters I taught that course was the great Mike Travisano.)

march-1986-working-late

October, 1986: Moving up in the world, we bought a new house on Mendel Court in University Hills. My parents visited, and that’s my mother, Jean, a poet who loved science.

october-1986-mendel-court-with-mom

March, 1987: The great Lin Chao came for a visit. We grew pea plants on the trellis below the number 6—after all, it was 6 Mendel Court.

march-1987-with-lin-chao

June, 1987: One of the fun events at UCI was Desert X (for extravaganza), hosted by Dick MacMillan, the chair of Ecology and Evolutionary Biology, on his property near Joshua Tree National Park. With Madeleine, who is “holding” our Number 3.

june-1987-desert-x-with-m

June, 1987: Working Xtra hard at Desert X with close friend and colleague Al Bennett.

june-1987-desert-x-with-al

September, 1987: With an already smiling one-month-old Natalie.

sept-1987-with-natalie

January, 1989: Time for some snuggles. Meanwhile, the LTEE is not quite a year old.

jan-1989-with-3-kiddos

The title of this post is a song by Fairport Convention, with the hauntingly beautiful voice of the late, great Sandy Denny. You should listen to it.

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Asking for a Skeptic Friend

I sometimes get email from people asking, in one way or another, whether our long-term evolution experiment (LTEE) with E. coli provides evidence of evolution writ large – new species, new information, or something of that sort. I try to answer these questions by providing some examples of what we’ve seen change, and by putting the LTEE into context. Here’s one such email:

Hi Professor Lenski,

I have a quick question. I’m asking because I am having a discussion with someone who is skeptical of evolution. The question is: Over the 50,000 generations of e-coli has any of the e-coli evolved into something else or is it still e-coli?

I am a non-religious person who likes to think of myself as an adherent to science but I am not sure how to respond to my skeptic-friend.

Thank you!

And here’s my reply:

Hello —-,

50,000 generations, for these bacteria, took place in a matter of ~25 years. They have changed in many (mostly small) ways, and remained the same in many other respects, just as one expects from evolutionary theory. Although these are somewhat technical articles, I have attached 3 PDFs that describe some of the changes that we have seen.

Wiser et al. (2013) document the process of adaptation by natural selection, which has led to the improved competitive fitness of the bacteria relative to their ancestors.

Blount et al. (2012) describe the genetic changes that led one population (out of the 12 in the experiment) to evolve a new capacity to grow on an alternative source of carbon and energy.

Tenaillon et al. (2016) describe changes that have occurred across all 12 populations in their genomes (DNA sequences), which have caused all of them to become more and more dissimilar to their ancestor as time marches on.

Best wishes,

     Richard

Although these articles were written for other scientists, they make three big points that I hope almost anyone with an open mind can understand.

  • We see organisms adapting to their environment, as evidenced by increased competitiveness relative to their ancestors.
  • Against this backdrop of more or less gradual improvement, we occasionally see much bigger changes.
  • And at the level of their genomes, we see the bacteria becoming more and more different from their ancestors.

In these fundamental respects, evolution in these flasks works in much the same way that evolution works in nature. Of course, the scales of time and space are vastly greater in nature than they are in the lab, and natural environments are far more complex and variable than is the simple one in the LTEE. But the core processes of mutation, drift, and natural selection give rise to evolution in the LTEE, just as they do (along with sex and other forms of gene exchange) in nature.

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A Birthday Sonnet

This past weekend, I celebrated my 60th birthday with friends and family from all over. One of the roasters was Ben “The Bard” Kerr, a professor at the University of Washington and colleague in the BEACON Center for the Study of Evolution in Action.

Borrowing from another bard, Ben waxed poetic about one of the lineages in the long-term evolution experiment and raised a toast with this Shakespearean flask.

 

Ben Kerr's Skakespearean flask

ODE TO AN LTEE LINEAGE

Shall I compare Ara-3 to a summer’s day?

Thou start more humbly, but sure potentiate.

Rough spins do shake the darling bugs of Rich’s gaze,

And latecomer’s “fleece” hath all to port citrate.

One line’s long-shot passed by eleven lines,

And how was its controlled complex “skin” pinned?

Promoter capture, over some time refined.

By chance, with nature’s arranging force, trimmed.

But thy Cit-minus partner shall not fade

Nor gain possession of the flair of most

C4 shall Cit snag, now spawned by carbon trade

Then on it turns ‘til lines will species now boast

     So long these cells can achieve, so wise to see,

     So long lives this work- and awe is rife, Lenski.

 

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Another Birthday Haiku

As I said in my last post, I just celebrated my 60th birthday with lots of friends and family. Several folks produced new artistic works, including two lovely haikus that celebrate the E. coli long-term evolution experiment.

Here’s one from Mike Wiser, who did his doctoral research on the long-term lines. A highlight of his work was a paper showing that fitness trajectories in these populations tend to follow a power law, which has no upper bound, rather than an asymptotic rectangular, as I had previously assumed.

Living things adapt.
Evolution keeps going.
No peak yet in sight.

 

Power law prediction, 2013

[The power-law model (blue) predicts future fitness gains much more accurately than does the hyperbolic model (red).  Image modified from Wiser et al. (2013, Science 342: 1364-1367) and shown here under the doctrine of fair use.]

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Birthday Haiku

This past weekend I had my 60th birthday. I was delighted to celebrate it with wonderful colleagues, students, friends, and family.

At a dinner roast and toast, everyone sang When We’re Sixty Four (Thousand), a tribute from the E. coli in the LTEE to the People of the Lab. And several friends came up with new contributions at the intersection of science and culture.

This beauty is from Andy Ellington, a professor in the Center for Systems and Synthetic Biology at the University of Texas and a member of the BEACON Center. As background, Andy coauthored a recent paper that helps to elucidate how one LTEE population evolved the novel ability to use citrate.

Without further ado, here’s his haiku …

Citrate just beyond.

Acetate potentiates.

Glucose is all gone.

 

Citrate

[Image of citrate molecule from Wikimedia Commons]

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Evolving Fun and Games

Science isn’t always fun and games. But sometimes it is!

This guest post is by Terry Soule, a computer scientist, and Barrie Robison, a biologist, both on the faculty at the University of Idaho. The BEACON Center for the Study of Evolution in Action brings together biologists, computer scientists, and engineers to illuminate and harness the power of evolution as an on-going process.

With BEACON’s support, Terry and Barrie have developed a video game, called Darwin’s Demons, where you must fight off enemies that are evolving to defeat your best efforts!

Feel free to comment here.  However, please send any technical queries via email to Terry (tsoule@cs.uidaho.edu) and/or Barrie (brobison@uidaho.edu).

*****

Thanks to BEACON’s support, Polymorphic Games has created the evolutionary video game Darwin’s Demons, and placed it on the Steam website as part of the greenlight process.

Darwin’s Demons adds an evolutionary component and modern flair to an arcade classic.  Darwin’s Demons models biological evolution using enemies with digital genomes. Enemies acquire fitness by being the most aggressive, accurate, and longest lived, and only the most fit enemies pass their genomes to the next generation. The result? The creatures you found hardest to kill have all the babies, making each generation more challenging than the last!

The game includes in-game graphs for tracking evolution, displays the most fit enemies from each wave, and has an experiment mode where you can set parameters like the mutation rate, fitness function, etc.  It also dumps all of the evolutionary data to a file.  So, there are opportunities for experiments on user driven evolution if anyone is interested.  (We are more than happy to share the code and/or make simple modifications for controlled experiments.)

If you get the opportunity please try out the demo (downloadable at either of the sites listed above, with Windows, MAC, and Linux versions), vote for us on Steam, and send us comments, suggestions, or ideas for future directions and collaborations.

Thanks,

— Terry Soule (tsoule@cs.uidaho.edu), Computer Science, UI

— Barrie Robison (brobison@uidaho.edu), Biological Sciences, UI

 

Darwin's Demons

[Darwin’s Demons: image from the Polymorphic Games website]

*****

 

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