Celebrating Black in STEM

I have been very fortunate to know and work with outstanding Black scientists throughout my career.  Here are a few of them.

I met Joe Graves when we were undergraduates at Oberlin College.  We took an evolution course together.  I remember discussing with Joe our mutual fascination with evolution and wondering how we might go about studying it.  I met up again with Joe at UC-Irvine, where we were both conducting evolution experiments—Joe using fruit flies, and me with bacteria.  Joe and I reconnected once more when he and I became founding members of the BEACON Center for the Study of Evolution in Action.  Joe now studies bacterial evolution, and we are becoming scientific collaborators as well.

JoAnn White was an ecologist at UNC, studying the life-history and population dynamics of periodical cicadas.  She served on my doctoral advisory committee and was a highly successful faculty member.  Unfortunately, she left academia, even though she had tenure, because it was too frustrating. I was honored that she asked me to write a reference letter when she moved to a new profession.  But it was a terrible loss for academia to lose such an outstanding scientist and role model as JoAnn White.

Paul Turner was one of my first graduate students.  He joined my lab in the Department of Ecology and Evolution at UC-Irvine, and he moved with me to MSU, receiving his Ph.D. in 1995.  Paul has impressed me in many ways, not only as a superb scientist and mentor, but also in his upbeat outlook on life.  Somehow he manages to smile and laugh about the challenges of being a departmental chair and interim dean, even while running a lab that conducts ground-breaking research.

Lynette Ekunwe was my lab manager and technician for seven years after I moved to MSU. She helped to sustain the long-term evolution experiment with E. coli after its move from UC-Irvine, and she helped run my lab group as it grew in size. Lynette moved to Jackson State University when her husband, the late Steve Ekunwe, took a faculty position there. After the move, Lynette earned a doctorate in public health, and she now works in the field of epidemiology.

I first met Scott Edwards when he was a graduate student at UC-Berkeley. I suspected that he was a rising star, and I was right.  Although Scott and I have not collaborated on actual science, we’ve worked together in other ways.  Scott and I served successive terms as Presidents of the Society for the Study of Evolution, and he has been a valued member of the External Advisory Board for the BEACON Center.

Shenandoah Oden was an undergraduate from Detroit when she joined my lab in the 1990s.  She worked with postdoc Santiago Elena on measuring the fitness effects of random insertion mutations in E. coli, leading to a paper in Genetica. What I remember best about Shenandoah is a question she asked me right after Brendan Bohannan presented his dissertation seminar: “How do scientists come up with the questions they ask?” I told her that was the best question that any student had ever asked me.  It reminded me of how Joe Graves and I, when we were undergrads, wondered how we might study evolution. To Shenandoah, I explained the importance of personal curiosity and mentors in finding questions that are both interesting and answerable.

Marwa Adewa, Maia Rowles and Kiyana Weatherspoon were three excellent undergraduate researchers in my lab, all of whom were mentored by Zachary Blount. Maia and Kiyana were coauthors on a paper in the Proceedings of the Royal Society, London B, which reported the results of what we call the “all-hands project”—one in which a generation of lab members performed a set of parallel assays to measure the subtle changes in fitness in late generations of the long-term evolution experiment with E. coli. Marwa now works in the field of veterinary medical research, while Maia and Kiyana are pursuing careers with a biomedical focus.

Judi Brown Clarke was, until very recently, the Diversity Director for our BEACON Center. In that role she generated and managed many successful programs that introduced hundreds of students at all levels to evolution and provided them with opportunities to engage in scientific research. She also was a great listener and valuable source of advice for many of us when we faced personal challenges and setbacks. An Olympic medalist, Judi recently became the Chief Diversity Officer at Stony Brook University.

I met Jay Bundy in 2013, at the Evolution meeting in Snowbird, Utah.  Who was this student who was asking so many thoughtful, insightful questions of the speakers?  I ran into Jay as we rushed between talks, introduced myself, and learned that he was a masters student at Penn State.  He wasn’t sure if he was interested in microbes, but I encouraged him to think about joining BEACON.  Jay came to MSU, first as a BEACON staff member contributing to education and outreach activities, and then as a graduate student in the Department of Integrative Biology.  He also contributed to the all-hands project.  However, he switched from studying bacteria to digital organisms, and he’s now performing and analyzing experiments to quantify how the duration of history in an evolving lineage’s previous environment influences its subsequent evolution in a new environment. Stay tuned for Jay’s findings—he’s working on a huge paper. Jay is as deeply thoughtful about science and life as I imagined when I first heard his questions at the Evolution meeting.

I also met Nkrumah Grant in 2013, when he visited MSU while exploring possible graduate programs.  He immediately impressed me with his personal story of overcoming obstacles.  Nkrumah explained to me his love of science as a child, and how he had gotten discouraged and derailed before undertaking a concerted effort to pursue his dream of science and scholarship.  And pursue it he did … and continues to do.  From a G.E.D. to a Ph.D.  Co-author on the all-hands project, co-first-author on a paper just published in eLife, and three more papers posted to bioRxiv in the last few weeks.  He also just defended his dissertation, giving a beautiful public seminar followed by an engaging, collegial exam.  Nkrumah has done all this and more while being a dedicated father and working tirelessly to promote equity and inclusion in science.

Last but not least, Ali Abdel Magid and Jalin Jordan are two of the current generation of superb undergraduate researchers in the lab. Ali is working with Nkrumah on the evolution of bacterial cell size, while Jalin works with Kyle Card on the evolution of antibiotic resistance.  Both Ali and Jalin are also working toward future careers in medicine. This summer, they are reading work that integrates evolution and medicine including the landmark book, Why We Get Sick, by Nesse and Williams, and the path-breaking paper by Tami Lieberman et al. on the evolution of bacteria in the lungs of CF patients.

My science is better, and my life richer, because of all these people, and many more.  How much better science would be, and how much richer all of our lives would be, if we would open more doors, listen more carefully, and live, learn, and work together.

***

After reading a draft of this essay, Nkrumah Grant, Joe Graves, and Jay Bundy all asked me to say more about this:  How can we achieve the aspirations expressed in my closing sentence above? 

I plan to reflect more on their vital question.  In the meantime, I invite readers who have ideas to put them in the comments below.

EDIT: I should also acknowledge two other influences: A high-school teacher, Mrs. Clayton, who taught a Black History class that I took, and who introduced me to Frederick Douglass, whose autobiography I read with awe and admiration.

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.