By JoVE, we’ve done it! – The Long-Term Evolution Experiment

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We recently published an open access video protocol in the Journal of Visualized Experiments (JoVE) covering some of the core procedures we use in the LTEE: the daily transfers, archiving the populations, and competition assays. It was a great experience discussing the details of these protocols with Zack, Devin, Minako, and Mike from Michigan State. Two of my lab managers at UT Austin, Emmanuel (former) and Jack (current) also helped a ton. Jack, in particular, stars in the video and had to put up with me behind the camera calling for take after take.

Let’s get another shot of that from the reverse angle!
It’s time for your close-up Ara–3!
The Director of the LTEE

Check out the video and the article if you want to learn about the LTEE or if you are considering starting your own microbial evolution experiment. The video is also embedded in this post below.

I’m looking forward to sharing these materials with new researchers joining my group who are learning to help maintain the LTEE or beginning to study it, and also with researchers we send these ever-evolving E. coli.

Some highlights:

We discuss aspects of the design of the LTEE that make it simple and sustainable.
We document all kinds of expected results: how the flasks appear after growth and the optical densities the evolved E. coli populations reach in different media; photos of the colonies formed by the evolved LTEE populations on various agar plates; and agar plates showing E. coli that form red and white colonies competing for dominance over several transfers and what the changes mean in terms of relative fitness values.
We created and an R package called fitnessR (available on GitHub) and an Excel spreadsheet (available as a supplemental file) for calculating relative fitness from co-culture competition experiment results.
We describe alternative procedures for performing competition assays that can be used when the fitnesses of the two competitors are very similar or very different.
We discuss some ways that the methods of the LTEE have been and are still being updated as technology has changed, and the potential advantages and disadvantages of other evolution experiment setups.

See also: If you are interested in LTEE methods, check out our protocols.io group which has media recipes and will gradually be populated with additional protocols. Rich also has a recent paper out that discusses the design of the LTEE, both in terms of procedures and as an experiment designed to answer questions about evolution (link).

Barrick, Jeffrey E.; Blount, Zachary D.; Lake, Devin M.; Dwenger, Jack H.; Chavarria-Palma, Jesus E.; Izutsu, Minako; Wiser, Michael J.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli Journal Article

Journal of Visualized Experiments, 198 , pp. e65342, 2023, ISSN: 1940-087X.

Abstract | Links | BibTeX

@article{Barrick2023,

title = {Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli},

author = {Jeffrey E. Barrick and Zachary D. Blount and Devin M. Lake and Jack H. Dwenger and Jesus E. Chavarria-Palma and Minako Izutsu and Michael J. Wiser},

doi = {10.3791/65342},

issn = {1940-087X},

year  = {2023},

date = {2023-08-18},

urldate = {2023-08-18},

journal = {Journal of Visualized Experiments},

volume = {198},

pages = {e65342},

abstract = {The Long-Term Evolution Experiment (LTEE) has followed twelve populations of \textit{Escherichia coli} as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions. },

keywords = {},

pubstate = {published},

tppubtype = {article}

}