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Evolution Experiments

Introduction

An evolution experiment consists of evolving one or more ancestor strains in laboratory culture and assessing the types and impacts of acquired mutations on the functions of that organism including its ability to reproduce, consume or produce compounds of interest, or resist particular stress conditions. The topics discussed on this page assume a general bacterial evolution experiment where a bacterial strain is serially passaged to fresh media over time. There are other types of evolution experiments including evolving phages, passaging pathogens and symbionts between hosts, and evolving multiple strains in co-culture that require additional considerations.

In general there are two kinds of evolution experiments: adaptive evolution and mutation accumulation. In adaptive evolution experiments cells within a culture compete with each other and trend towards an increase in fitness. These experiments are useful for identifying mutations that improve a particular function, such as growth on a particular compound or resistance to a particular stress. Individuals with detrimental mutations are usually out-competed, thus the mutations evolved in these experiments is usually biased towards beneficial mutations. Mutation accumulation experiments, on the other hand, reduce or remove selection, allowing the persistence of all types of mutations and are thus useful for calculating overall mutation rates or examining the spectrum of possible mutations. The effect of selection is usually limited by selecting colonies to streak from one plate to another at randon. Since colonies are grown initially from individual cells this also represents the smallest possible bottleneck size. The discussion on this page applies predominantly to adaptive evolution experiments, you can read more about mutation accumulation experiments here.

Experimental design

An overall evolution experiment will likely involve three phases: 1) inoculating populations, 2) routine transfers, and 3) isolating clones and freezing stocks.

1) Inoculating populations Regardless of whether they are adaptive or mutation accumulation experiments, evolution experiments should be started by streaking the ancestral stock onto an agar plate and then initiating each population from a separate isolated colony. This reduces the impact of mutations that may already be present in the ancestral stock and ensures that the populations begin as single genotypes. Also set up a separate tube or flask with an uninoculated control to check for contamination. After the first day of growth populations should be frozen so they can be sequenced and referred to later, in particular when determining whether identical mutations shared between samples evolved during the experiment or were present in the initial culture.

2) Routine transfers Over the course of an evolution experiment the populations will be transferred to fresh media to continue growing after their initial media has been used up. Designing an effective evolution experiment requires taking into account the impact of:

• Volume of transfers
• Timing of transfers Cultures should be transferred at regular intervals after they have reached saturation, usually every 24 hours (+/- one hour). Particularly in the beginning of an experiment, before they've adapted to the culture conditions, it's possible some populations will not reach saturation in one day. It's usually best to postpone transferring all the cultures for an additional day to allow them all to reach saturation and remain on the same transfer schedule.
• Number of generations
• Stress conditions If the goal is to improve resistance to a particular stress condition it may be beneficial to increase the stress over time as the strains adapt to increasing concentrations.
• Back-up cultures After transferring cultures to fresh media, the previous day's cultures should be stored at 4°C until the next day. If an uninoculated control shows contamination the back-up cultures should be removed from the fridge, warmed up, vortexed, and transferred from instead. Note that this does not count as progressing the total number of generations again as the previous day's transfer was unsuccessful.

3) Isolating clones and freezing stocks At the end of the transfer period the evolved strains should be frozen for further analysis. For an adaptive evolution experiment this can be done as populations by freezing culture media from the last transfer and/or as isolates by streaking each population on a plate and inoculating a large, isolated colony into fresh media and then freezing it. Isolates can also taken from frozen populations after the experiment by thawing and streaking out the stock.

Analyzing evolution experiments

The end result of an evolution experiment is likely isolated, evolved clones that have acquired additional mutations relative to their ancestor. The goals, then, are usually to identify these mutations in the genome and determine what their functional consequences, if any, are. These can be accomplished by sequencing the isolates' genomes and comparing them to the ancestor with breseq and performing fitness assays or other functional assays (i.e. growth curve, minimum inhibitory concentration, biochemical assays, as appropriate), respectively.