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]

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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!

We Interrupt This Experiment

Today I made the decision to close the lab and temporarily suspend our experiments, including the LTEE, in light of the expanding SARS-CoV-2 outbreak.

I started to say it was a difficult decision, but really it was not all that difficult.  Several considerations led me to this decision.

1/ The SARS-CoV-2 outbreak appears to be taking off in many countries, including the USA, despite the substantial containment that has been orchestrated in Wuhan and elsewhere in China.

2/ The absence of evidence of any local cases is not as comforting as it might be, given the near-absence of testing here and in most of the USA.

3/ MSU students just returned from spring break today.  Some of the superb undergrads who work in my lab went to places that have confirmed cases. None of the places they went are among the locations with intense outbreaks, but the confirmed cases in at least one location have grown noticeably in the past week. They also flew on planes to and from their vacations.

4/ As a team, we’re connected not only to one another, but also to people who are health-care workers and others with increased vulnerabilities to infections. (Not to mention that I’m over 60 …) When you think about it, pretty much everyone has those connections.

5/ We’re very lucky because our work is easy to stop and re-start. Our study organisms can be frozen away and revived whenever we see fit.  In the meantime, everyone has classes to take, papers to read and write, data to analyze, etc.  And a little extra time, hopefully, to reflect on and maintain the health and well-being of our friends, families, and selves.  So, we will all be busy, but doing things a bit differently than we had planned.

6/ As we freeze away the long-term lines, the lab notebook will record:  “On this day, the LTEE was temporarily halted and frozen down for the coronavirus pandemic of 2020.”

Hopefully, some future historian of science will look back on today’s entry and say: “What the hell was that all about?”

[Devin Lake putting the LTEE populations into the ultra-low freezer, where they will stay until they are called back into action … evolution in action.]

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.]

 

Thank you, Neerja

Neerja Hajela has worked with me for over 22 years as a technician and lab manager. She is extremely skillful, diligent, organized, and dedicated in her work. On top of all that, she is a wonderfully kind and warm person. Now, this is her last week before she retires.

It’s impossible to put into words all that Neerja has done for me, for everyone in the lab, for the LTEE, and for my collaborators. But let me mention a few of the things she has done.

By keeping the lab running in a smooth and orderly fashion, Neerja has enabled me to spend more of my time thinking about science, writing papers, giving talks, etc., etc. We scientists sometimes complain that we have too much to do, and so we joke that we want to clone ourselves. Well, I’ve done better—I’ve had Neerja.

Those of us with labs know that our institutions take laboratory safety very seriously, as well they should. Neerja runs such a tight ship that, on many occasions after inspecting our lab, the safety officers have made comments to the effect that they wish all labs were as neat, clean, organized, and safety-conscious as ours.

One of the challenges of the long-term evolution experiment (LTEE) with E. coli is freezer management. We now have over 30 years of samples, spread over half a dozen freezers, which provide a record of past evolution. Neerja has overseen this ever-growing collection with extraordinary care and dedication. The samples provide critical backups that allow us to restart the LTEE from a recent milestone when mishaps occur, and they provide unique research materials such as when new technologies emerge. A case in point: Michael Desai wrote me a few years ago with a request. In essence, he wanted all of the LTEE samples for metagenomic sequencing. All of them—from each population and every generation with saved samples. Since I started the LTEE in 1988, we’ve always saved duplicate samples, with one of them being a backup to be opened only in an emergency. I could send Michael the backups, perhaps, but that didn’t seem like a good idea. So I decided we should make additional sets by going into the ~1500 key samples spread over several freezers; taking a subsample of each and culturing it to produce a larger sample; splitting the new culture into ten sub-cultures; and freezing those to provide a new set for Michael as well as other sets for collaborators and institutions. It took Neerja many months to accomplish all of this, but as always, she did it with great skill and care. (Oh, and you can read about the results of Michael’s request here.)

Last, but surely not least, Neerja has done more of the daily transfers of the LTEE than anyone else. She performed her first LTEE transfer on February 5, 1996, and since then she has done well over 4,200 daily transfers. (Thanks to Zachary Blount, who went through the LTEE lab notebooks for its 30^th birthday.) And when Neerja hasn’t done the transfers herself, she has organized who else is responsible for each and every day’s transfers.

Thank you, Neerja, for all that you have done for me, for everyone in the lab, for the LTEE, and for science. Everyone in the lab joins me in wishing you and Ravindra all the very best in your retirement and new home!

 

[Neerja Hajela]

[Here’s Neerja doing yesterday’s LTEE transfer]

[Neerja pointing at two of her LTEE entries: her first transfer on February 5, 1996, and the one from yesterday July 30, 2018. The lab notebooks in the background record the daily transfers since she joined the lab.]

[Close-up of Neerja’s first entry.]

[Neerja’s protocols for the LTEE, mounted in the lab, so nobody makes a mistake]

[And in case that doesn’t work, here’s Neerja enforcing lab discipline]

[The LTEE transfer board from earlier this year]

[Horsing around Tanush’s plate tower]

[Neerja making copies of freezer samples]

[Neerja and me]