The E. coli long-term evolution experiment (LTEE''') is an ongoing study in experimental evolution begun by Richard Lenski at the University of California, Irvine, carried on by Lenski and colleagues at Michigan State University, and currently overseen by Jeffrey Barrick at Michigan State University. It has been tracking genetic changes in 12 initially identical populations of asexual Escherichia coli bacteria since 24 February 1988. Lenski performed the 10,000th transfer of the experiment on March 13, 2017. The populations reached over 73,000 generations in early 2020, shortly before being frozen because of the COVID-19 pandemic. In September 2020, the LTEE experiment was resumed using the frozen stocks. When the populations reached 75,000 generations, the LTEE was transferred from the Lenski lab. In August 2024, the LTEE populations passed 80,000 generations in the Barrick lab.
Over the course of the experiment, Lenski and his colleagues have reported a wide array of phenotypic and genotypic changes in the evolving populations. These have included changes that have occurred in all 12 populations and others that have only appeared in one or a few populations. For example, all 12 populations showed a similar pattern of rapid improvement in fitness that decelerated over time, faster growth rates, and increased cell size. Half of the populations have evolved defects in DNA repair that have caused phenotypes marked by elevated mutation rates. The most notable adaptation reported so far is the evolution of aerobic growth on citrate, which is unusual in E. coli, in one population at some point between generations 31,000 and 31,500. However, E. coli usually does grow on citrate in anaerobic conditions and has an active citric acid cycle which can metabolize citrate even under aerobic conditions. The aerobic event is mainly an issue of citrate being able to enter the cell.
On May 4, 2020, Lenski announced a five-year renewal of the grant through the National Science Foundation's Long-Term Research in Environmental Biology (LTREB) Program that supports the LTEE. He also announced that Jeffrey Barrick, an associate professor of Molecular Biosciences at The University of Texas at Austin, would take over supervision of the experiment within the five-year funding period. The experiment's time at Michigan State University ended in May 2022, when the populations reached 75,000 generations but the experiment was revived and restarted in Barrick's lab on June 21, 2022.
In 2025, Barrick was hired by the Michigan State University Department of Microbiology, Genetics, and Immunology as a Hannah Distinguished Professor. In August of that year, his lab moved to MSU, bringing the LTEE back to Michigan.
Experimental approach The long-term evolution experiment was designed as an open-ended means of empirical examination of central features of evolution. The experiment was begun with three principal goals: To examine the dynamics of evolution, including the rate of evolutionary change; To examine the repeatability of evolution; To better understand the relationship between change on the phenotypic and genotypic levels. As the experiment has continued, its scope has grown as new questions in evolutionary biology have arisen that it can be used to address, as the populations' evolution has presented new phenomena to study, and as technology and methodological techniques have advanced.
The use of E. coli as the experimental organism has allowed many generations and large populations to be studied in a relatively short period of time. Moreover, due to the long use of E. coli as a principal model organism in molecular biology, a wide array of tools, protocols, and procedures were available for studying changes at the genetic, phenotypic, and physiological levels. The bacteria can also be frozen and preserved while remaining viable. This has permitted the creation of what Lenski describes as a "frozen fossil record" of samples of evolving populations that can be revived at any time. This frozen fossil record allows populations to be restarted in cases of contamination or other disruption in the experiment, and permits the isolation and comparison of living exemplars of ancestral and evolved clones. Lenski chose an E. coli strain that reproduces only asexually, lacks any plasmids that could permit bacterial conjugation, and has no viable prophage. As a consequence, evolution in the experiment occurs only by the core evolutionary processes of mutation, genetic drift, and natural selection. This strict asexuality also means that genetic markers persist in lineages and clades by common descent, but cannot otherwise spread in the populations.
Lenski chose to carry out the experiment with the bacteria grown in a glucose-limited medium called DM25, which is based on a minimal medium developed by Bernard Davis for use in isolating auxotrophic mutants of E. coli using penicillin as a selective agent. To make DM25, the minimal medium is supplemented with a low concentration (25 mg/L) of glucose. Lenski chose this concentration to simplify analysis of the populations' evolution by reducing clonal interference, in which multiple versions of alleles are competing in an evolving population, while also reducing the possibility of the evolution of ecological interactions. This concentration of glucose used supports a maximum population of 500 million cells of the ancestor in a 10 mL culture, though the maximum now varies among the evolved populations. DM25 also contains a large amount of citrate (about 11 times the concentration of glucose), which was originally included by Davis because it improved the killing efficiency of penicillin during his experiments, though it is now known to aid in E. colis acquisition of iron from the medium.
Methods The 12 populations are maintained in a incubator in the Barrick laboratory at University of Texas at Austin. Each day, 1% of each population is transferred to a flask of fresh DM25 growth medium. The dilution means that each population experiences 6.64 generations, or doublings, each day. Large, representative samples of each population are frozen with glycerol as a cryoprotectant at 500-generation (75-day) intervals. The bacteria in these samples remain viable, and can be revived at any time. This collection of samples is referred to as the "frozen fossil record", and provides a history of the evolution of each population through the entire experiment. The populations are also regularly screened for changes in mean fitness, and supplemental experiments are regularly performed to study interesting developments in the populations. , the E. coli populations have been under study for over 64,500 generations, and are thought to have undergone enough spontaneous mutations that every possible single point mutation in the E. coli genome has occurred multiple times.
Founding strain The strain of E. coli Lenski chose to use in the long-term evolution experiment was derived from "strain B", as described in a 1966 paper by Seymour Lederberg (which incorrectly identified the strain as "Bc251", although later genetic analysis found it to be "B" instead), via Bruce Levin, who had used it in a bacterial ecology experiment in 1972. The defining genetic traits of this strain were: T6r, Strr, r−m−, Ara− (unable to grow on arabinose). Lenski designated the original founding strain as REL606. Before the beginning of the experiment, Lenski isolated an Ara+ variant of the strain in which a point mutation in the ara operon had restored growth on arabinose, which he designated as strain REL607. When beginning the long-term evolution experiment, Lenski founded six populations with six individual Ara− colonies of REL606. These populations are referred to as Ara-1 through Ara-6. Lenski also founded six more populations from six individual Ara+ colonies of REL607. These are referred to as populations Ara+1 through Ara+6. The marker differences permit strains to be differentiated on Tetrazolium Arabinose plates, on which Ara− colonies appear red, while Ara+ colonies appear white to pink. Over the course of the experiment, each population has accumulated a large number of distinct mutations, which permit further means of identifying strains by their population of origin.
Results Changes in fitness
Much analysis of the experiment has dealt with how the fitness of the populations relative to their ancestral strain has changed. All populations showed a pattern of rapid increase in relative fitness during early generations, with this increase decelerating over time. By 20,000 generations the populations grew approximately 70% faster than the ancestral strain. This increase and deceleration in increase has continued in subsequent generations. A 2013 study by Wiser et al. reported ongoing improvement at 50,000 generations relative to samples isolated at 40,000 generations. They found that the fitness increase fit to a power law model much better than the hyperbolic models that had been used earlier. As a power law model describes an ever-slowing increase that has no upper limit, while a hyperbolic model implies a hard limit, the work suggested that the increase would continue without bound as progressively lower benefit mutations were fixed in the populations. Further work published in 2015 reported the results of over 1100 new fitness assays that examined fitness changes through 60,000 generations. The data once again fit the proposed power law model, and, indeed, fit within predictions of the model from earlier data. These results suggest that, contrary to previous thinking, adaptation and adaptive divergence can potentially increase indefinitely, even in a constant environment.
Genome evolution Of the 12 populations, six have so far been reported to have developed defects in their ability to repair DNA, greatly increasing the rate of mutation in those strains. Although the bacteria in each population are thought to have generated hundreds of millions of mutations over the first 20,000 generations, Lenski has estimated that within this time frame, only 10 to 20 beneficial mutations achieved fixation in each population, with fewer than 100 total point mutations (including neutral mutations) reaching fixation in each population. In 2009, Barrick et al. reported the results of genome sequences from multiple time points in population Ara-1. They found that, unlike the declining rate of fitness improvement, mutation accumulation was linear and clock like, even though several lines of evidence suggested that much of the accumulation was beneficial, rather than neutral.
Evolution of increased cell size in all twelve populations
All twelve of the experimental populations show an increase in cell size concurrent with a decline in maximum population density, and in many of the populations, a more rounded cell shape. This change was partly the result of a mutation that changed the expression of a gene for a penicillin-binding protein, which allowed the mutant bacteria to outcompete ancestral bacteria under the conditions in the long-term evolution experiment. However, although this mutation increased fitness under these conditions, it also increased the bacteria's sensitivity to osmotic stress and decreased their ability to survive long periods in stationary phase cultures.
Ecological specialization