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.