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

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.