Inside a 37-Year Journey with E. coli
In a laboratory in Texas, twelve populations of bacteria have been evolving for over 80,000 generations, revealing profound truths about how life adapts.
Explore the ExperimentImagine an experiment that watches evolution unfold in real-time, tracking genetic changes across thousands of generations without pause. This isn't science fiction—it's the E. coli Long-Term Evolution Experiment (LTEE), launched in 1988 by biologist Richard Lenski and still running today under Jeffrey Barrick's supervision at the University of Texas at Austin 2 .
What began as an investigation into evolutionary dynamics has transformed into one of biology's most remarkable long-term studies, producing insights that reshape our understanding of adaptation, mutation, and the very tempo of evolutionary change.
Richard Lenski designed the LTEE with three fundamental questions in mind: How predictable is evolution? At what rate do evolutionary changes occur? And how do changes in organisms' physical traits relate to underlying genetic mutations? 2
The researchers chose a special strain of E. coli that reproduces asexually, lacks plasmids, and has no viable prophage 2 .
The team regularly freezes samples with glycerol as a cryoprotectant, creating a complete history of evolution 4 .
Each day, researchers transfer 1% of each population into fresh DM25 growth medium 4 .
The heartbeat of the LTEE is its daily transfer ritual, a carefully choreographed procedure that has continued with few interruptions since 1988 4 . Each day, researchers transfer 1% of each population into fresh DM25 growth medium—a glucose-limited solution where the concentration of glucose (25 mg/L) is substantially lower than that of citrate (1.7 mM), which is included as an iron chelator 2 9 . This 1:100 dilution allows the bacteria to undergo approximately 6.64 generations (doublings) each day before the glucose is exhausted and they enter stationary phase 3 .
| Reagent/Equipment | Function in the Experiment |
|---|---|
| DM25 Growth Medium | Defined glucose-limited medium with only 25 μg/mL glucose, forcing competition for limited resources 2 3 |
| Disodium Citrate | Iron chelator in DM25 medium; originally included for penicillin enrichment 2 9 |
| Glycerol | Cryoprotectant used to preserve population samples in the "frozen fossil record" 4 |
| Tetrazolium Arabinose (TA) Agar | Specialized agar that differentiates Ara⁻ (red colonies) and Ara⁺ (white colonies) strains 2 4 |
| 50 mL Erlenmeyer Flasks | Culture vessels that provide consistent aeration and growth conditions 4 |
One of the most important measurements in the LTEE is relative fitness—how well the evolved bacteria compete against their ancestors. Researchers measure this using co-culture competition assays 3 4 . In these assays, they mix an evolved population (usually Ara⁻) with an ancestral competitor (Ara⁺) and allow them to grow together for one day under the same conditions as the LTEE 3 .
The populations are distinguished by plating samples on TA agar, where Ara⁻ and Ara⁺ cells produce red and white colonies respectively 4 . By counting the colonies at the beginning and end of the competition, scientists can calculate exactly how much the ratio has changed, generating a precise measurement of relative fitness.
Every 75 days (approximately 500 generations), researchers preserve the evolutionary history of each population by archiving samples 4 . This process involves:
Taking a representative sample from each population
Mixing with glycerol as a cryoprotectant
This "frozen fossil record" serves multiple purposes: it provides material for future studies, allows the experiment to be restarted from any point if contamination occurs, and enables direct comparisons between ancestral and evolved clones 2 4 . The preservation of this detailed history has allowed scientists to use technologies that didn't exist when the experiment began—including whole-genome sequencing—to analyze evolutionary changes 4 .
One of the most fundamental findings from the LTEE concerns the pattern of fitness improvement over time. Early in the experiment, fitness increased rapidly, but the rate of improvement decelerated over time 2 .
By 20,000 generations, the populations grew approximately 70% faster than their ancestor 2 . Surprisingly, this improvement has continued without stopping. A 2013 study found that fitness increases followed a power law model, suggesting that adaptation can potentially continue indefinitely, even in a constant environment, as progressively smaller beneficial mutations continue to accumulate 2 .
Perhaps the most dramatic event in the LTEE occurred between 31,000 and 31,500 generations in one population (Ara-3), when the bacteria evolved the ability to consume citrate under aerobic conditions 2 .
This was remarkable because E. coli typically cannot grow on citrate in the presence of oxygen, and citrate was present in the growth medium throughout the experiment as an iron chelator 2 9 . This innovation wasn't a single mutation but required multiple genetic changes that eventually allowed citrate to enter the cell and be metabolized 2 .
| Evolutionary Change | Populations Affected | Significance |
|---|---|---|
| Improved competitive fitness | All 12 populations | Pattern of rapid improvement that decelerated over time, with cells growing ~70% faster than ancestor by 20,000 generations 2 |
| Increased cell size | All 12 populations | All populations showed increased cell size and, in many cases, a more rounded cell shape 2 |
| Elevated mutation rates | 6 of 12 populations | Defects in DNA repair led to mutation rates 100-1,000 times higher than non-mutator lines 2 |
| Citrate utilization (Cit⁺) | 1 population (Ara-3) | Evolution of aerobic growth on citrate, a previously unused resource in the medium 2 |
| Citrate dependence | 3 populations (besides Ara-3) | Evolved dependence on citrate as an iron chelator for optimal growth on glucose 9 |
| Genetic Change Type | Scale of Change | Notable Examples |
|---|---|---|
| Point mutations | 10-20 beneficial mutations fixed in each population in first 20,000 generations 2 | Mutations in topA and fis genes affecting DNA supercoiling |
| Insertion sequence movements | Multiple insertions in each population | Insertions affecting metabolic genes 6 |
| Deletions | Loss of functions not needed in environment | Loss of rbs operon preventing growth on ribose 6 |
| Structural variations | Inversions and other rearrangements | Various chromosomal rearrangements |
After more than 80,000 generations and 37 years, the LTEE continues to yield new insights. The experiment survived a potentially disastrous interruption—the COVID-19 pandemic—by using its frozen fossil record to restart after a temporary freeze 2 . In 2022, the experiment moved from Michigan State University to the University of Texas at Austin when Jeffrey Barrick took over leadership 2 .
Richard Lenski initiates the LTEE with twelve identical populations of E. coli 2
Populations grow approximately 70% faster than their ancestor 2
One population evolves the ability to consume citrate under aerobic conditions 2
Study reveals fitness increases follow a power law model 2
Experiment moves to University of Texas at Austin under Jeffrey Barrick's supervision 2
Experiment continues beyond 80,000 generations, with new technologies extracting fresh discoveries 6
New technologies continue to extract fresh discoveries from this long-running experiment. Recent research has integrated genomic data with metabolic profiling to understand how mutations affect cellular chemistry 6 . This work provides steps toward a complete genotype-phenotype map—a fundamental goal in biology that would allow us to predict how genetic changes will affect an organism's traits and fitness 6 .
The LTEE has become a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology 1 4 . Its robust methods have served as a template for countless new evolution experiments with different microbes, environments, and questions 4 . Samples from the frozen fossil record are now studied on every continent except Antarctica by researchers younger than the experiment itself 4 .
The Long-Term Evolution Experiment demonstrates the relentless power of natural selection even in the simplest environments. What began as a straightforward investigation into evolutionary dynamics has become an invaluable window into the fundamental processes that shape life.
The twelve populations of E. coli, initially identical, have diversified in surprising ways, revealing both the predictable patterns and creative possibilities of evolution.
As the experiment continues, it promises further insights into life's endless capacity for innovation. The LTEE stands as a testament to the value of long-term research and the unexpected discoveries that can emerge from patiently watching evolution unfold, one bacterial generation at a time.