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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.


[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.


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


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.)


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.


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.


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


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


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


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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At my 60th birthday party this summer, I made a few remarks about how fortunate I have been in my life:

Born to parents who nurtured me.

Born into a nation that values life, liberty, and the pursuit of happiness.

Born at a time and in a part of the world where science and public health greatly improved my chances of survival and good health. (Living to age 60 was once a rarity, and it still is in much of the world.)

Fortunate to have had a superb education, and to have met so many wonderful people along the way, including my wife.

Lucky to have three talented, interesting, and kind children, two loving and good sons-in-law, and now two healthy grandkids.

Fortunate to have a career where I get to study how the world works, and where I get to work with incredibly talented and motivated students and colleagues.

Today I was reminded of another aspect of privilege:

Privilege is getting to vote with no long lines and without intimidation. I was privileged today. I wish all Americans had that privilege.

It’s something we should all embrace.  Working to deny citizens their right to vote is wrong. It also threatens all of us today and future generations, and the freedoms and privileges that we sometimes take for granted.

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When We’re Sixty Four (Thousand)

From the E. coli in the LTEE to the People of the Lab

[To be sung along to this Beatles classic]


When we get older, losing our fimbriae,

Many years from now,

Will you still be sending us our thiamine,

Birthday greetings, Erlenmeyer wine?

If we were mutants, crazy and fit,

Would that make you snore?

Will you still feed us, will you still freeze us,

When we’re sixty-four?


You’ll be older too,

And if you say the word,

We’ll evolve with you.


We could be handy, helping your pubs,

When your grants are gone.

You can write a paper by the fireside,

Weekend days give no time to hide.

Colonies growing, dotting the plates,

Who could ask for more?

Will you still feed us, will you still freeze us,

When we’re sixty-four?


Every summer you can buy a freezer when the space gets tight,

If it’s not too dear.

Save our clonal mix,

Plus and minus progeny,

Ara One to Six.


Keeping the notebook, pipetting each drop,

Track trajectories.

Indicate precisely what you think will change.

Hypothesize, test, unlimited range.

Give us your data, sequence and store,

Evolving evermore.

Will you still feed us, will you still freeze us,

When we’re sixty-four?



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A Life Well Lived, Part II

This second tribute to my father was written by my son Daniel, who gave me permission to post it here.

~~~ ~~~ ~~~

My dear Grandpa Gerry died yesterday at 91, at home near Seattle with Grandma Ann and three of my aunts by his side. He had a sharp and curious mind undimmed by age, and a kind and sympathetic ear despite his deafness. He enjoyed many years of good health, and I particularly remember his smile after he kept pace with my father during a long walk up a sand dune, in his late 70s.

Grandpa was born and raised in Washington, DC at a time when slabs of ice were delivered in horse-drawn carts, and kids could freely roam the White House grounds and all the embassies, sneak up into the Capitol dome, and surreptitiously feed bubblegum to monkeys through the bars at the National Zoo. I hope the statute of limitations on that particular incident has run out.

As a cryptographer in WW2, Grandpa encoded messages with geared machines weighing hundreds of pounds, surrounded by walls lined with dynamite, yet he also lived long enough to get the hang of touchscreens, to print out this XKCD cartoon and tape it to the side of his iMac, and most importantly to Skype with his great-grandchildren. He got a DNA profile done, and seemed kinda bummed to find out that he was probably not descended from Genghis Khan. In his career as a sociologist he studied religion and technology and critiqued totalitarian governments (topics as important as ever today), wrote several books, and figured out how to edit his own Wikipedia page. I remember more than once in recent years when I stayed up late talking about my life and work at Intel with Grandpa, only to find that he had woken up before me the next morning, brimming with new questions and ideas.

He was an old dog still learning new tricks. On October 30 we went to his favorite restaurant. I drove, but Grandpa pointed out all the shortcuts in the dark. When the waitress came by to take our drink orders, I expected he’d get his usual deer-in-the-headlights look and blurt out “Bud Light,” at which point I’d protest and order him something more interesting. But this time was different. Without missing a beat, Grandpa set down his menu, asked for a Mac and Jack Amber Ale, and turned to me silently with a twinkle in his eye.

~~~ ~~~ ~~~

My son Daniel, me, and my father Gerry in 2012

My son Daniel, me, and my father Gerry in 2012

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A Life Well Lived

My father died peacefully at his home near Seattle this morning, before dawn, at 91 years of age. Gerhard Emmanuel Lenski, Jr. was born (1924) and raised in Washington, DC. His father went by Gerhard, and my father went by “Gerry” (pronounced like Gary) his whole life. My father did his undergraduate and graduate studies at Yale University, with his undergraduate years interrupted by three years of service with the US Army Air Forces during World War II, most of which was spent in England as a cryptographer at a joint USAAF-RAF airbase.

After receiving his Ph.D. in 1950, my father joined the Department of Sociology at the University of Michigan, where he rose through the faculty ranks. In 1963, he moved to the University of North Carolina, where he was Alumni Distinguished Professor and served as department chair for several years. He retired in 1992. He wrote several important books including “The Religious Factor: A Sociological Study of Religion’s Impact on Politics, Economics, and Family Life” (1961), “Power and Privilege: A Theory of Social Stratification” (1966), “Ecological-Evolutionary Theory: Principles and Applications” (2005), and “Human Societies: An Introduction to Macrosociology” (1970), now in its 12th edition (2014). He served as vice president of the American Sociological Association, and as president of the Southern Sociological Society. His honors included a Guggenheim Fellowship, election to the American Academy of Arts and Sciences, and a Career of Distinguished Scholarship Award from the American Sociological Association.

In 1948, my father married my mother, the former Jean Cappelmann, a poet, and they had 4 children. They were active together in working for civil rights and against the Vietnam War. They were married for 45 years before my mother passed away in 1994. In 1996, my father married the former Ann Blalock, who was a close family friend and whose late husband Hubert “Tad” Blalock, had been a colleague of my father’s at both the University of Michigan and the University of North Carolina.

After moving to the Seattle area, my father enjoyed visiting northwest sites and cities including the Olympic National Park, Mount Baker, Portland (where my son lives), and Victoria; cheering on the Seahawks and Mariners; watching the ships on the Puget Sound; and talking with his children and grandchildren, always full of questions and ideas about technology and life.

My father was beloved by family and friends for his storytelling and humor – who can forget the story about the time he and a childhood friend gave their chewing gum to monkeys at the National Zoo? – as well as his deep knowledge of and appreciation for human history.

My father was fortunate to have lived a good and full life for 91 years, and I was very lucky to have him for almost six decades. I was also lucky to spend Thanksgiving with him, and we had the chance to share many stories that spanned his life—from baseball trivia to meeting his newest great-grandson in my father’s first-ever Skype.

Dad and Me on Dad's 90th

My father and me on his 90th birthday

ADDITION 1: Click here for a picture of my father from his days at UNC.

ADDITION 2: My son Daniel wrote a wonderful tribute to his Grandpa here.


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63,000 Strong

Ever wonder about those big numbers posted in a window in that tall building on the east side of Farm Lane, across from the entrance to the MSU Dairy Store?

Right now, the digits read 63000. That’s the number of generations in an experiment that’s been running in my lab for over a quarter century.

We call it the LTEE, which stands for the Long-Term Evolution Experiment. There are 12 populations of E. coli bacteria in the experiment, and they all started from the same strain.

Every day—weekends and holidays included—a member of my team takes 1% of the cells in each population and puts them in a flask with fresh food. Over the next 24 hours, the population grows 100-fold and then runs out of food. These dilutions and renewals go on day after day, week after week, month after month, year after year, decade after decade. I hope the experiment will continue long after I’m gone, so that someday someone can write “and century after century.”

Bacteria grow by binary fission: 1 cell makes 2 cells, 2 cells make 4, 4 make 8, etc. So the 100-fold growth in the fresh medium represents about 6.6 doublings, or generations, every day. (There’ve been some interruptions since the LTEE began in 1988, but not many.)

Now consider a bacterial cell that gets a mutation in its DNA that lets it acquire more food and grow a little faster. That cell will leave more descendants than its competitors—that’s adaptation by natural selection. Over time, the bacteria are becoming stronger and fitter in their flask-worlds.

By watching the 12 populations evolve, we can answer questions about the dynamics and repeatability of evolution in a group of organisms—bacteria—that are essential for life on Earth as well as important players in health and disease. We measure the growth rates of the bacteria, we sequence their DNA, and we see just how much evolution can achieve even in short order.

Oh, about the sign. Zachary Blount is a talented postdoc who works on this project, and he likes to have fun with science. He put up the window display which, if you look closely, has a picture of Charles Darwin on the left, “The E. coli Long-Term Evolution Experiment” over the number, and “Generations and Counting” to the right. Every 1,000 generations or so, Zack updates the sign.

63K window

[Photo credit: Zachary D. Blount]

Note:   This piece first appeared at after an invitation from Alice Dreger to explain the numbers in the window to our community.

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