We thank Richard Lenski and the many researchers who have studied and contributed to maintaining the Long-Term Evolution Experiment with E. coli, including especially Neerja Hajela. The LTEE is currently supported by the National Science Foundation (DEB-1951307).

No conflicts of interest declared.

Long-term resilience of the LTEE and its methods 
The E. coli Long-Term Evolution Experiment (LTEE) is now in its fourth decade. For a microbial evolution experiment of any duration, it is critical to maintain a reproducible environment, avoid contamination, archive samples, and accurately measure fitness. The LTEE demonstrates several time-tested strategies for achieving these objectives, including the use of well-shaken flasks that create a homogenous environment and a chemically defined growth medium that supports a low cell density. Moreover, the LTEE employs ancestor strains that differ in a genetic marker that gives a phenotype (colony color) that is both easily screened and selectively neutral in the evolution environment. This experimental design feature provides a means of identifying internal and external contamination and facilitates measuring fitness. However, not all of the procedures and safeguards that have been used by the LTEE since 1988 have proven equally robust. Some methods that were reliable when the LTEE began have become less effective as the E. coli populations have evolved. Fortunately, these problematic methods can now be augmented or replaced using technologies developed since the experiment&#39;s inception.
Detecting contamination 
Detection of contamination is critical to the LTEE. Contamination can be of two sorts: between LTEE populations (cross-contamination) and with microbes from the environment (outside contamination). For the most part, careful use of aseptic techniques and close attention during media preparation and the daily transfers prevent both types of contamination, but they do happen. Early in the experiment, plating on TA agar could be used to detect instances of cross-contamination because transfers have always alternated between Ara&#8722; and Ara+ populations. The fingerprint of sensitivity and resistance of these E. coli to certain bacteriophages was also intended to be a design feature that could differentiate the LTEE populations from commonly used E. coli laboratory strains that might contaminate them4. However, these genetic markers have become unreliable as the experiment has progressed (e.g., some populations no longer form colonies on TA agar)10,35. Fortunately, the populations have genetically diverged as they have experienced separate evolutionary histories during the experiment, which has created new genetic markers that can now be used to detect cross-contamination. For instance, each population has evolved a unique combination of mutations in the pykF and nadR genes14,36,37. We sometimes PCR amplify and Sanger sequence these two genes to test whether colonies with unusual morphologies or colors are due to cross-contamination. As the costs of whole-genome and whole-population sequencing continue to drop, routine sequencing of the LTEE populations may soon be possible, thereby presenting new opportunities to monitor them for signs of contamination.
Measuring competitive fitness 
Another case in which the LTEE has outgrown its original methods is that the fitness of the evolved E. coli has increased in the experimental environment to such a degree that one can no longer directly measure the fitness of the populations of today relative to their ancestors using the protocol described here. The evolved populations outcompete the ancestors to such an extent that few to no ancestor colonies remain to count after a one-day competition. One approach for dealing with this large fitness difference is to use unequal starting ratios of the strains, weighting the initial volumes that are mixed toward the less-fit competitor (e.g., 90 &#181;L ancestor and 10 &#181;L evolved competitor). A second approach is to identify an evolved Ara&#8722; clone that has a higher fitness than the LTEE ancestor, isolate a spontaneous Ara+ revertant mutant of it by selection on MA agar, and then verify that the revertant strain has the same fitness as its parent using a competition assay6,38. This new Ara&#8722;/Ara+ pair can then be used as a set of common competitor strains in lieu of REL606/REL607. Ideally, the evolved Ara&#8722; clone chosen as a common competitor (and its Ara+ revertant) will have intermediate fitness relative to all strains of interest in an experiment. Over the first 50,000 generations of the LTEE, these two approaches (using unequal starting ratios or a common competitor) did not produce meaningfully different fitness measurements versus the typical approach39.
These modifications to the competition protocol make certain simplifying assumptions that may not always be true. One is that fitness measurements are transitive. That is, if we compete two populations each versus a common competitor strain separately, then we can infer the relative fitness of the two populations to one another. This relationship has been found to be true for the LTEE40, for the most part, but it is not for other experiments41. One reason for this discrepancy can be the evolution of negative frequency-dependent fitness effects. This situation occurs when strains isolated from two different diverged lineages from population A&#8722;2 of the LTEE are competed against one another19,42. Each has an advantage when rare, due to cross-feeding, which stabilizes their co-existence. Sequencing data showing long-term co-existence of lineages with different sets of mutations suggests that similar interactions may also have arisen in other LTEE populations14,43, though it is not clear whether they are strong enough to noticeably alter fitness estimates. Finally, the evolution of aerobic growth on citrate in population A&#8722;3 of the LTEE32 means that the fitness of these cells now incorporates the use of a &#34;private&#34; resource when they are competed against cells that cannot use citrate, which complicates interpreting these results. Despite these exceptions, the use of a low glucose concentration and well-shaken environment has undoubtedly simplified making fitness comparisons between LTEE strains and populations.
At later generations, some of the LTEE populations no longer form colonies on TA agar, which makes performing competition experiments using even modified protocols difficult or impossible10. Alternative methods that do not require colony growth can potentially be used to determine the relative representation of two competitors, such as FREQ-seq which uses next-generation sequencing to count the proportion of reads containing two alternative alleles in an amplicon44. This method or a similar one could potentially be used with the Ara alleles or with newly evolved mutations, such as those in pykF and nadR, versus the ancestral sequence. Performing genetic modifications that introduce other types of neutral markers can also be used to measure relative fitness. For example, fluorescent protein genes have been inserted into the chromosomes of cells in LTEE offshoot experiments so that competitors can be counted using flow cytometry45. Another approach, which opens up the possibility of mixing together more than two strains in the same competition flask, is to insert barcodes that can be PCR amplified and sequenced into the genomes of different competitors. This approach has been used for lineage tracing in evolution experiments46. Both flow cytometry and barcode sequencing can accurately measure much more extreme ratios of two strains versus colony counting (because they can query &#62; 10,000 cells/genomes versus the &#60; 500 that can be counted on an agar plate), so using these methods also promises to increase the dynamic range in terms of fitness differences that can be measured relative to a common competitor.
Alternative designs for long-term microbial evolution experiments 
For all its virtues, the LTEE is not perfect. Certain aspects of its design make it labor intensive and susceptible to human error. For instance, each day a researcher must come into lab and pipette between Erlenmeyer flasks to continue the experiment. Competition experiments can also pose daunting logistical hurdles, given that requirements for sterile glassware, media, incubator space, and colony counting rapidly escalate when even a small number of competitors are being tested with modest replication. We are often asked why we don&#39;t take advantage of laboratory automation systems, such as pipetting robots that operate on 96-well microplates, or continuous culture systems, such as chemostats or turbidostats. The answer is simple: the LTEE is, in a sense, a prisoner of its own long history. We dare not deviate from 10 mL&#160;cultures shaking at a specific speed in 50 mL Erlenmeyer flasks because this would risk fundamentally changing the experiment. Subtle aspects of the environment to which these populations have been adapting for decades (e.g., the amount of aeration), would be altered in microplates or continuous culture systems. The population bottleneck at each transfer might also be different (smaller in microplates, for example), changing the evolutionary dynamics. In short, deviating from the methods described here would make the LTEE a different experiment, or at the very least risk introducing a discontinuity that would disrupt evolutionary trajectories.
Researchers designing new evolution experiments should consider these other ways of propagating microbial populations, while being aware of their potential benefits and drawbacks. Using pipetting robots to transfer populations in microwell plates is logistically simpler in some ways and can prove quite powerful due to the high numbers of replicate populations that can be propagated in this way47,48,49. However, automated transfers in most current setups do not take place under completely sterile conditions, which increases the likelihood of outside contamination. To prevent contamination, the growth medium is often supplemented with antibiotics, which become a feature of the environment that affects evolution. Transfers in microwell plates are also more prone to cross-contamination events. Finally, the environment of microwell plates&#160;&#8212; particularly if they are not shaken&#160;&#8212; tends to select for wall growth, aggregation, and other phenomena that can complicate evolution by creating multiple niches in one well. Using rich media or high concentrations of nutrients to keep population sizes large in small wells is likely to exacerbate these complexities. If such interactions arise, they can make measuring and interpreting fitness much more difficult.
Continuous culture systems for microbial evolution include chemostats, in which fresh medium&#160;is constantly pumped in and culture is pumped out, and turbidostats, in which cultures are periodically diluted through automated sensing and pumping to maintain cells in a state of constant growth. These systems are very useful when one wants to model microbial physiology and evolution because they avoid having microbes transition between growth and starvation by keeping them in an environment that always has nutrients50. One can even add sensors that make real-time measurements of optical density, O2 consumption, pH, and other aspects of a culture&#39;s environment and growth. However, current continuous culture systems either require expensive equipment purchases or specialized expertise to build custom setups51,52,53,54. Also, wall growth, in which cells escape dilution by adhering to the culture chamber, bedevils evolutionary dynamics in continuous culture systems unless they are periodically sterilized. Due to these constraints, most chemostat and turbidostat evolution experiments to date have been of limited duration and/or involved relatively few independently evolving populations compared to serial transfer evolution experiments.
Conclusion 
The methods we demonstrate here for the LTEE are critical for studying its unique historical record and continuing the open-ended evolution of these E. coli populations. They also provide a starting point for others who are considering new evolution experiments that may take advantage of laboratory automation or add back various elements of the complexity found in natural environments that were purposefully omitted from the LTEE. Since 1988, experimental evolution has flourished as a field. During this time, researchers in laboratories across the globe have demonstrated the immense flexibility of this approach for studying evolution, innovating by introducing creative experimental designs and monitoring the results using new technologies. The methods of the LTEE do not represent an endpoint, but we hope they will continue to inspire and provide a foundation for the field far into the future.

In February of 1988, Richard Lenski inoculated twelve flasks containing a defined glucose-limited growth medium with clonal cultures of Escherichia coli at the University of California, Irvine1. The following day, he transferred 1% of the culture from each flask to a set of new flasks containing fresh growth medium. This 1:100 dilution allowed the bacterial populations to expand 100-fold before exhausting the available glucose, corresponding to roughly 6&#8532; generations of cell divisions. This procedure was repeated the following day and has been every day since, with a few interruptions. These daily transfers have continued, even as the experiment was relocated, first to Michigan State University in 1992, and then to The University of Texas at Austin in 2022. All the while, new mutations have continuously generated genetic variation in these E. coli populations and natural selection has led to evolved cells outcompeting their ancestors.
Lenski designed this experiment, now known as the Long-Term Evolution Experiment (LTEE), to investigate the dynamics and repeatability of evolution. In order to answer these questions, he included several important features in the design of the experimental setup and its protocols2. One of these features was the careful choice of a model organism. The original twelve populations were all started from single colonies that shared an immediate common ancestor, Escherichia coli B strain REL606. This strain was chosen because it had already been commonly used in lab settings, reproduced completely asexually, and contained no plasmids or intact prophages3,4 &#8212; all of which make studying its evolution simpler. Another choice that simplified the experiment was to use a very low concentration of glucose in the growth medium to limit the density of cells in each flask after growth. Using a low cell density was intended to make it easier to analyze changes in population fitness by reducing the potential for the evolution of ecological interactions within populations (e.g., by cross-feeding)5.
REL606 is unable to use &#671;-arabinose as a carbon and energy source (Ara&#8722;) due to a point mutation in the araA gene. Prior to starting the LTEE, a spontaneous mutant with a restored araA sequence, designated REL607, was isolated from REL6066. REL607 is capable of growing on &#671;-arabinose (Ara+). REL606 was used to start six of the LTEE populations, and REL607 was used to start the other six. Arabinose is not present in the growth medium used during the LTEE, so REL607 behaves the same as REL606 under these conditions. However, when plated on tetrazolium arabinose (TA) agar, Ara&#8722; and Ara+ cells form red and white colonies, respectively. This method for discriminating between the two ancestral E. coli strains and their descendants is quite useful. It can be used to detect cross-contamination between LTEE populations. It also aids in measuring the fitness of an Ara&#8722; strain or population relative to an Ara+ one when they are competed against one another. Fitness is measured by setting up a co-culture of oppositely marked competitors and then monitoring how the frequencies of red and white colonies (obtained by spreading dilutions of the culture on TA plates) change between when the competitors are initially mixed and after one or more growth cycles under the same conditions as the LTEE. The representation of the more-fit cell type will increase during each growth cycle.
Another critical feature of the LTEE is that samples of the evolving populations are periodically archived. When mixed with a cryoprotectant such as glycerol, E. coli cells can be frozen and later revived7. As part of the LTEE protocol, every 75th day (which equates to roughly 500 generations), a portion of each population that was not transferred to a new flask is mixed with glycerol, split up between multiple vials, and stored in a freezer. This frozen &#34;fossil record&#34; enabled researchers to perform the first studies of the LTEE, in which they revived the evolved E. coli populations from various timepoints and competed them versus the ancestral strains to track how rapidly fitness was increasing1. Fitness evolution has been remeasured periodically as more &#34;strata&#34; of the frozen &#34;fossil record&#34; have been preserved. The overall conclusion from these measurements is that fitness continues to improve in the LTEE to this day, even after so many generations of evolution in the same environment8,9,10.
What has allowed the LTEE to continue for so long? Many of the same features that enabled its original questions to be asked and answered have also served as safety measures and fail-safes against inevitable disruptions due to bad luck, human error, and world events. Every day, when the cultures are transferred to&#160;fresh growth medium, the researcher performing the transfers alternates between Ara&#8722; and Ara+ populations. Then, when the populations are frozen, they can be plated on selective and indicator agar to check if any &#34;neighboring&#34; populations have been accidentally cross-contaminated or mixed up (e.g., white colonies are in a population that should only form red colonies) or contaminated with foreign microbes (e.g., unexpected colony morphologies or cell densities). In the event that a population has been compromised, its progenitor can be revived from the freezer and carried forward in its place. The Ara markers and the frozen archive thus serve dual purposes as both experimental resources and safety measures.
Because its history is so well preserved and easily accessible, LTEE samples have been studied using technologies that did not exist when the experiment began. For example, whole-genome sequencing has been used to examine the dynamics of mutations in the LTEE populations11,12,13,14,15, and transcriptomics and ribosomal profiling have been used to examine changes in gene expression16,17. Genetic tools have been used to reconstruct strains that differ by single mutations or combinations of several evolved mutations to understand their effects on fitness and various phenotypes18,19,20,21. Samples from the frozen &#34;fossil record&#34; are easily replenished so that parts of or entire copies of the experiment&#39;s history can be shipped to other laboratories. LTEE samples now exist on all continents except Antarctica, and they are being studied by researchers who are younger than the experiment itself. The robust methods of the LTEE and evolved E. coli samples and strains from its historical record have also served as starting points for evolution experiments examining other questions and environments22,23,24,25,26,27,28,29.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65342/65342fig01v2.jpg" /> 
Figure 1: Overview of LTEE procedures. <a href="https://www.jove.com/files/ftp_upload/65342/65342fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we demonstrate three core protocols used in the E. coli Long-Term Evolution Experiment (Figure 1). We describe: (1) how to perform the daily transfers, (2) how to archive population samples and clonal isolates, and (3) how to perform and analyze co-culture competition assays to measure fitness differences. Our hope is that these protocols foster the continued use of LTEE resources and inform the design of new microbial evolution experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride (TTC)</td>
			<td>Sigma-Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Glass Beaker</td>
			<td>Sigma-Aldrich</td>
			<td>CLS100020</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Erlenmeyer Flasks</td>
			<td>Sigma-Aldrich</td>
			<td>CLS498050</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>AX1385</td>
			<td></td>
		</tr>
		<tr>
			<td>Antifoam</td>
			<td>Sigma-Aldrich</td>
			<td>A5757</td>
			<td></td>
		</tr>
		<tr>
			<td>Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Box (2&#34;)</td>
			<td>VWR</td>
			<td>82007-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Box (3&#34;)</td>
			<td>VWR</td>
			<td>82007-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Box Cell Divider (49-place)</td>
			<td>VWR</td>
			<td>82007-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Box Cell Divider (81-place)</td>
			<td>VWR</td>
			<td>82007-154</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Vials (1/2-Dram)</td>
			<td>VWR</td>
			<td>&#160;66009-816&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Vials (2-Dram)</td>
			<td>VWR</td>
			<td>&#160;66010-560&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal Tray</td>
			<td>Winco</td>
			<td>SPJP-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Tribasic Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7254</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Tube Cap (18mm)</td>
			<td>VWR</td>
			<td>10200-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Tube Rack (18mm, steel)</td>
			<td>Adamas-Beta</td>
			<td>N/A</td>
			<td>Test Tube Racks Stainless Steel Grid Arrangement 72 Holes (17-19 mm)</td>
		</tr>
		<tr>
			<td>Test Tubes (18 x 150 mm)</td>
			<td>VWR</td>
			<td>47729-583</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine, Hydrochloride</td>
			<td>Millipore</td>
			<td>5871</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Gibco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Gibco</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

daily transfers, archiving populations, and measuring fitness in the long-term evolution experiment with escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of 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.

Watch this Scientific Journal Video about Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli at JoVE.com

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

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

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

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

In 1988, Richard Lenski began an evolution experiment. He filled 12 Erlenmeyer flasks with a glucose-limited defined growth medium known as DM25. He inoculated six of these flasks with an E.coli strain known as REL606 which is unable to utilize the sugar arabinose as a nutrient.These populations were designated A-1 through A-6. He inoculated the other six with a nearly identical strain known as REL607 which is able to utilize arabinose. These ancestral strains, the E.coli that evolved from them, can be distinguished when plated on tetrazolium or arabinose agar.The Ara-strains form red colonies, and the Ara+strains form white colonies. Lenski placed these flasks in a shaking incubator at 37 degrees Celsius, allowing them to grow to saturation and exhaust the glucose in the medium. At this point, he transferred 1%of the culture from each of the flasks into a new set of 12 flasks containing fresh medium.These daily cycles of transfer and regrowth have been continued for decades, creating a long history of evolution that can be studied by researchers. In our protocol, we describe how the long-term evolution experiment, or LTEE, populations are transferred and cultured each day. Then we describe how the populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen record for later study.We often refer to this as the, quote, fossil record of the LTEE. An important way that the overall tempo and character of evolutionary changes is monitored in the LTEE is through measuring the relative fitnesses of populations and strains from the experiment. We describe how to conduct these co-culture competition assays and analyze the resulting data to calculate relative fitness values.We'll show the first and the last of these procedures in this video. First, we'll demonstrate a daily transfer of the long-term evolution experiment. Disinfect the surface on which the LTEE transfers will be conducted by wiping it with either 70%ethanol or a 10%bleach solution.Light a Bunsen burner to create a local updraft and enable the flaming of glassware. Prepare 13 50-milliliter Pyrex Erlenmeyer flasks capped with 20-milliliter Pyrex or polypropylene beakers that have been washed and sterilized by autoclaving. Check the flasks for visible debris and replace any that are not perfectly clean.Label six flasks A-1 through A-6 using a red marker, and the other six flasks A+1 through A+6 using a black marker. Label the last remaining flask, which will be the blank, with the date, in month-day format, and the day of the week. Fill each of the 13 flasks with 9.9 milliliters of DM25 medium using a sterile 10-milliliter serological pipette.Flame the mouth of each flask after removing the beaker serving as a lid, and before replacing the beaker. Flame the tip of the pipette between filling each flask. Remove the prior day's LTEE flasks from the shaking incubator.Examine each in turn by holding it up to the light to assess its turbidity and color. Check flask integrity and look for the presence of foreign matter. Using a P200 micropipettor with a sterile filter tip, transfer 100 microliters of culture from each LTEE flask into the corresponding flask containing fresh DM25.Begin with A-1, then transfer A+1. After that, continue alternating between the minus and plus populations. To keep track of which cultures have been transferred, shift flasks to the left as pipetting from or to them.Observe strict aseptic technique during transfers. Use a fresh pipette tip for each transfer. Flame the mouths of flasks immediately after uncapping and before recapping, and wipe down the barrel and injector of the micropipettor with a Kimwipe moistened with 70%ethanol between each transfer.Incubate the newly inoculated flasks at 37 degrees Celsius for 24 hours with 120 revolutions-per-minute orbital shaking over a one-inch diameter. Store the cultures from the previous day at four degrees Celsius. Retain these cultures for two days.Discard older cultures that were saved at four degrees Celsius three days before at this time. Enter the time, date, transfer number, your name or initials, whether or not the cultures were okay, and any other relevant information in the transfer log notebook. Another 6 2/3 generations have passed in the history of the long-term evolution experiment with E.coli.If there are any problems, accidents, or suspicions of contamination with the previous day's LTEE cultures, do not transfer from them. Instead, store the entire set of 12 cultures at four degrees Celsius for later examination and further characterization. Retrieve the backup flasks that were transferred from the day before and stored at four degrees Celsius.Place them on the benchtop to warm to room temperature. Gently swirl each flask to resuspend the cells. Transfer from each flask to fresh medium in a new set of flasks and continue the experiment normally.Make a note in the transfer log that you used the backup cultures and record the same transfer number as the day before. If contamination is noted in the backup flasks stored at four degrees Celsius, then the affected LTEE populations must be restarted from frozen stocks. Next, we'll demonstrate a co-culture competition assay that can be used to measure the relative fitness of two strains or populations from the long-term evolution experiment.We'll be showing a competition experiment that evolves both of the LTEE ancestors, REL606 and 607, and two evolved populations, population A-5 at 20, 000 generations, REL8597;and population A+5 at 20, 000 generations, REL8604. These assays used sixfold replication for each pair of an Ara-and Ara+competitor. Decide how many competitor LTEE strains and/or populations you'll be using and how many replicate competition assays will be performed for each pair of competitors.Prepare the necessary supplies as follows. For the revival day, day minus two, fill one sterile 50-milliliter Erlenmeyer flask capped with a 20-milliliter beaker with 9.9 milliliters of either DM1000 or lysogeny broth, LB, per strain or population of E.coli that will be used as a competitor. Fill one more flask with 9.9 milliliters of the same medium to serve as an uninoculated blank.For the preconditioning day, day minus one, fill one test tube with 9.9 milliliters of 0.85%weight-per-volume sterile saline per competitor, two flasks with 9.9 milliliters DM25 per replicate assay between a pair of competitors, and one more flask with 9.9 milliliters of DM25 for a blank. For the day the competition begins, day zero, fill one flask with 9.9 milliliters of DM25, fill one test tube with 9.9 milliliters of sterile saline, and prepare one tetrazolium or arabinose, or TA plate, per competition assay replicate. Fill one more flask with 9.9 milliliters of DM25 to serve as a blank.If you are performing a three-day competition, fill two additional sets of competition flasks. For the final day of the competition, fill two test tubes with 9.9 milliliters of sterile saline and prepare one TA plate per competition replicate. On day minus two of a competition experiment, you revive the competitors separately from freezer stocks.Take the cryovials containing the frozen stocks of the competitor strains out of the 80 degree Celsius freezer. Keep the vials chilled in a nice bucket while using them. After each frozen stock thaws, vortex it thoroughly to resuspend the E.coli cells.If reviving a clone, inoculate the flask containing fresh medium with 12 microliters of the frozen stock. If reviving a population, inoculate with 120 microliters of the frozen stock. Incubate the revival flasks in the blank at 37 degrees Celsius overnight with 120 revolutions-per-minute orbital shaking over a one-inch diameter.On day minus one of a competition experiment, you precondition the competitors separately in DM25. Take the flasks containing the cultures of revived competitors out of the incubator. Transfer 100 microliters from each flask into the test tube of saline for that competitor.Vortex each dilution tube thoroughly right before transferring 100 microliters from the diluted culture into a flask with fresh DM25. Inoculate two of these preconditioning flasks for each replicate assay, one for each of the competitors. Incubate the preconditioning flasks in the blank at 37 degrees Celsius for 24 hours.On day zero of a competition experiment, you begin the competition by mixing the competitors and plate for initial counts. Take the preconditioning flasks out of the incubator. Transfer 50 microliters of the Ara-competitor into the first replicate competition flask filled with fresh DM25.Immediately transfer 50 microliters of the Ara+competitor into the same competition flask. Mix the flask by gently swirling. Repeat these steps to combine all replicates of all pairs of competitors you were testing.Transfer 100 microliters from each newly inoculated competition flask into the test tube of saline labeled for that competition assay replicate. Place the new competition flasks and blank into the shaking incubator. Meanwhile, on the same day, vortex each test tube thoroughly and plate 80 microliters of these 10, 000-fold dilutions on TA plates.Label the side of the bottom of each plate with the pair of strains that were mixed, the replicate number, and day zero to indicate it will be used to determine the initial representation of each competitor. Incubate the TA plates upside down in a gravity convection incubator at 37 degrees Celsius until colonies of both the Ara-and Ara+competitors are visible and distinguishable. Generally, this occurs within 16 to 24 hours, but it may take longer for some evolved strains.Count the numbers of Ara-red, and Ara+white, colonies on each plate and record the results. If you are performing a three-day competition, transfer 100 microliters of each competition replicate to 9.9 milliliters of fresh DM25 medium in a new flask after growth. Incubate these day-one flasks for 24 hours.Perform another transfer in the same way and incubate the day-two flasks for 24 hours to complete three full co-culture growth cycles of the competitors under LTEE conditions. On the final day of the competition, day one or day three, depending on the length of your experiment, you'll plate for final counts. Take the competition flasks out of the incubator.Transfer 100 microliters from each competition flask into the first test tube of saline for that replicate. Vortex each 100-fold dilution tube to mix it thoroughly and transfer 100 microliters to the second tube of saline for that replicate. The resulting tubes contain 10, 000-fold dilutions of the DM25 cultures.Vortex each test tube containing a 10, 000-fold dilution thoroughly and plate 80 microliters of it on a TA plate. Label the side of the bottom of each plate with the pair of strains that were mixed, the replicate number, and day one for a one-day competition or day three for a three-day competition to indicate that it will be used to determine the final representation of each competitor. Incubate TA plates at 37 degrees Celsius and count the Ara-and Ara+colonies after growth.Use the Excel spreadsheet provided with this protocol, or the Fitness RR package, to calculate the relative fitness of the strains in your competition and plot the results. During the daily transfers of the long-term evolution experiment, the density of the cultures after growth is very low and must often be judged by holding a culture up right next to a blank. The exception is population A-3 that has evolved to use citrate in the medium as an additional nutrient source and grows to about tenfold the cell density of the other populations.Measurements of the optical density in the LTEE cultures can also be used to monitor for expected growth. For the example competition assays, we expect a more fit competitor to increase its representation over time, relative to a less fit competitor. Examining the TA plates with colonies grown from dilutions of just one replicate of the competition between the two ancestor strains shows that the ratio remains relatively constant over the course of a three-day competition experiment.By contrast, the A+5 population rapidly outcompetes the REL606 ancestor. And the A-5 population rapidly outcompetes the REL607 ancestor. The two evolved populations are nearly evenly matched.The representations of red and white colonies remain almost the same over the three days of the competition. Using the colony counts after one day of co-culture, we find that the fitnesses of the evolved populations have increased by 60%during the LTEE relative to the ancestral strains of E.coli. By continuing to transfer the competitions over multiple days, we can improve the precision of relative fitness measurements for competitors that are closely matched.But when two competitors have very different fitnesses, there is no longer an adequate representation of both colonies after a multi-day competition to calculate the relative fitness. The methods we demonstrate here are critical for studying the unique historical record of the LTEE and for continuing this open-ended evolution experiment. They can also provide a starting point for others who are considering new evolution experiments that address new questions, use new environments, and study different microorganisms.

daily-transfers-archiving-populations-measuring-fitness-long-term

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

Abstract