In Other News

Today is the 34th birthday of the LTEE, which I started on February 24, 1988. 

With the invasion of Ukraine, however, it’s not a day to celebrate.

 The LTEE will move to the capable lab and hands of Jeff Barrick this Spring, after all 12 lines have reached 75,000 generations.

Over the decades, several lines fell behind others due to cross-contamination (or concerns about the possibility), which we detected by examining the alternating Arabinose marker and seeing the resulting colony colors on TA plates. Those lines were then restarted from whole-population samples, but they would be 500 generations behind the others (or a multiple of 500 generations behind in some cases).

The picture above shows red and white colonies growing on TA agar in a Petri dish. The red colonies cannot grow on the sugar arabinose that is part of the TA medium, while the white ones can use arabinose. Half of the LTEE lines started from red colonies (Ara–1 to Ara–6), and half started from white colonies (Ara+1 to Ara+6). We alternate the red and white lines each day during their propagation. That way, if cross-contamination occurs, we can detect it by the presence of bacteria that make colonies that are the wrong color. We check colonies before every periodic freeze of the LTEE. These days, with DNA sequencing, we can also use derived mutations that are unique to each lineage to check whether a putative contamination event is real or not. (Indeed, in some populations, especially those that evolved hypermutability, the colony markers don’t work like they did when the LTEE started.) If we confirm that a cross-contamination event has occurred, we restart the affected population from the last frozen sample of that population.

So today, Devin Lake will propagate the last two lagging populations. Our lab will continue to propagate them until they, too, reach 75,000 generations. The last one should reach that goal in late May.

They’re back!

They’re back! After a six-month interruption, Devin Lake restarted the long-term evolution experiment and the 12 LTEE lines from the 73,000-generation freezer samples. Now the bacteria are back in their home-sweet-homes: Erlenmeyer flasks with DM25 medium and the shaking incubator set to 37C.

We’re keeping the lab at very low occupancy, and using masks and physical distancing when more than one person is present in a room) until this damn SARS-CoV-2 pandemic is under control.

MSU also has a spit-based surveillence program in place for those entering campus buildings. Each sample is split, and then put into two pools for PCR testing. With each individual’s sample split into two pools, the testing can identify which individual in any reaction that proves to be positive is the source of the virus. That person is then notified and told to isolate and get a definitive diagnostic test.

[Both photos below courtesy of Devin Lake]

Five More Years

The E. coli long-term evolution experiment (LTEE) began in 1988, and it has run for over 32 years with only occasional interruptions. The latest interruption, of course, reflects the temporary closure of my lab during the ongoing coronavirus pandemic. Fortunately, one of the advantages of working with bacteria is that we can freeze population samples and later revive them, which will allow us to resume their daily propagation when it is prudent to do so.  Indeed, we’ve frozen samples of all 12 populations throughout the LTEE’s history, allowing “time travel” to measure and analyze their fitness trajectories, genome evolution, historical contingencies, and more.

Even as the experiment is on ice, the lab team continues to analyze recently collected data, prepare papers that report their findings, and make plans for future work. Their analyses use data collected from the LTEE itself, as well as from various experiments spun off from the LTEE.  Nkrumah Grant is writing up analyses of genomic and phenotypic aspects of metabolic evolution in the LTEE populations.  Kyle Card is examining genome sequences for evidence of historical contingencies that influence the evolution of antibiotic resistance. Zachary Blount is comparing the evolution of new populations propagated in citrate-only versus citrate + glucose media. Minako Izutsu is examining the effects of population size on the genetic targets of selection, while Devin Lake is performing numerical simulations to understand the effects of population size on the dynamics of adaptive evolution.  So everyone remains busy and engaged in science, even with the lab temporarily closed.

Today, I’m excited to announce two new developments.  First, the National Science Foundation (NSF) has renewed the grant that supports the LTEE for the next 5 years. This grant enables the continued propagation of the LTEE lines, the storage of frozen samples, and some core analyses of the evolving populations. The grant is funded through the NSF’s Long Term Research in Environmental Biology (LTREB) Program, which “supports the generation of extended time series of data to address important questions in evolutionary biology, ecology, and ecosystem science.” Thank you to the reviewers and program officers for their endorsement of our research, and to the American public and policy-makers for supporting the NSF’s mission “to promote the progress of science.”

Second, Jeff Barrick joins me as co-PI on this grant for the next 5 years, and I expect he will be the lead PI after that period.  In fact, Jeff and his team will take over the daily propagation of the LTEE populations and storage of the sample collection even before then. I’m not planning to retire during the coming grant period. Instead, this transfer of responsibility is intended to ensure that the LTEE remains in good hands for decades to come. In the meantime, Jeff’s group will conduct some analyses of the LTEE lines even before they take over the daily responsibilities, while my team will continue working on the lines after the handoff occurs.

Several years ago I wrote about the qualifications of scientists who would lead the LTEE into the future: “My thinking is that each successive scientist responsible for the LTEE would, ideally, be young enough that he or she could direct the project for 25 years or so, but senior enough to have been promoted and tenured based on his or her independent achievements in a relevant field (evolutionary biology, genomics, microbiology, etc.). Thus, the LTEE would continue in parallel with that person’s other research, rather than requiring his or her full effort, just like my team has conducted other research in addition to the LTEE.”

Jeff is an outstanding young scientist with all of these attributes. Two years ago he was promoted to Associate Professor with tenure in the Department of Molecular Biosciences at the University of Texas at Austin.  He has expertise in multiple areas relevant to the LTEE including evolution, microbiology, genomics, bioinformatics, biochemistry, molecular biology, and synthetic biology. He directs a substantial team of technicians, postdocs, and graduate students, which will provide ample coverage for the daily LTEE transfers (including weekends and holidays). Last but not least, Jeff has participated in the LTEE and made many contributions to it including:

• Participated in propagating the LTEE lines and related activities while he was a postdoc in my lab from 2006 to 2010.
• Authored many papers using samples from the LTEE, including almost all of them that have analyzed genome sequences as well as several recent papers examining the genetic underpinnings of the ability to use citrate that evolved in one lineage.
• Developed the open-source breseq computational pipeline for comprehensively identifying mutations that distinguish ancestral and evolved genomes.

Someone might reasonably ask if the LTEE will work in the same way when it is moved to another site. The answer is yes: the environment is simple and defined, so it is readily reproduced. Indeed, I moved the LTEE from UC-Irvine to MSU many years ago, the lab has moved between buildings here at MSU, and we’ve shared strains with scientists at many other institutions, where measurements and inferences have been satisfactorily reproducible. As an additional check, Jeff’s team at UT-Austin ran a set of the competition assays that we use to measure the relative fitness of evolved and ancestral bacteria, and we compared the new data to data that we had previously obtained here at MSU. The two datasets agreed well, in line with the inherent measurement noise in assessing relative fitness. Fitness is the most integrative measure of performance of the LTEE populations, and it is potentially sensitive to subtle differences in conditions. These results provide further evidence that, when the time comes, the LTEE can continue its journey of adaptation and innovation in its new home.

Evolve, LTEE, evolve!

Freezer Burn

One of the more challenging aspects of running a microbiology lab, in my opinion, is freezer management.  There’s a lot to keep track of, both in terms of quantity and quality.  My lab team and I take great pride in the quality control of our work that has allowed us, for example, to keep the LTEE running for over 30 years and 70,000 generations without contamination.  Or rather, as I’ve posted before, we’ve had occasional accidents including cross-contamination of the replicate lines, but we’ve caught those mistakes and, using frozen samples, restarted as needed to keep things going smoothly and cleanly.

With my lab group now running for ~34 years (I started at UCI in 1985), and with so many hard-working students and postdocs, we’ve filled up lots of –80C freezers.  And that’s despite shipping many strains to scientific collaborators and former lab members who’ve continued to work on the various projects—the LTEE is only one (albeit the longest) of the many projects we’ve done in my lab.  Adding to the storage challenge, we’ve got duplicates of most samples in case we have a problem with the primary sample (say, someone drops a vial on the floor).  Also, to avoid compromising our primary or backup samples, I ask that everyone who plans to use any sample (usually a set of many samples) more than once make his or her own working copies of the samples.

And freezers sometimes fail, despite our best efforts to maintain them in tip-top shape.  So over the years, I’ve always tried to keep a freezer’s worth of spare capacity across our multiple freezers, so when one fails, everything can be moved into a functioning freezer.

On Sunday, one of our workhorse freezers failed. Most of our freezers have alarms that send out an email alert to members of the lab that something is amiss.  This one did not (oops!), but fortunately undergraduate Jessica Baxter (working hard even on the weekend), noticed that it had “warmed up” to –40C or so.  I was off visiting grandkids, but Jessica was able to reach Devin Lake, who manages the lab’s operations extremely well, even as he does double-duty as a grad student.  Devin and Jessica were able to find enough spare capacity to get everything into one of the surviving freezers, so nothing was lost.

But that meant we had no more spare capacity.  We can buy a new freezer, although my experience (and hearing about many other failures) is that they don’t make them like they used to.  And what if another freezer were to fail before we got a new one?

I knew we had many freezer racks full of now-unimportant samples—working copies made by people who’ve left the lab, as well as samples from abandoned experiments and various long-ago projects that won’t be revisited.  So I asked Devin to look through the freezers for the identifiers on various racks (besides the LTEE and any associated with current lab members) that would give me ideas of what we could discard to free up some space that we will need for ongoing projects … as well as the possibility of another freezer failure.  (But please not that!  I’m not trying to tempt fate—I just want to be prepared.)  It turns out there were lots of possibilities, so Devin and I spent a couple of hours checking boxes and then removing about 20 freezer racks, most holding 6 to 10 boxes, and most of those with dozens of small vials, each holding many millions or even billions of bacterial cells.  Seeing the names of former lab members on the boxes, and the numbers on all those vials, was a humbling reminder of all the hard work that so many have done over the years.  Devin carted three loads of discards down to one of our workrooms, where hardworking tech John Baltusis emptied each box and prepared the vials for the sterilization (autoclaving at high temperature) that’s required before they can be discarded.

Thanks to the hard work of Jessica, Devin, and John, the lab avoided any setback. In fact, our freezer collection is now a little more manageable than it was before.

[Devin Lake, in front, with one of three cartloads of samples to discard, while John Baltusis removes the samples from one box before autoclaving.]

 

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!!