Understanding that evolution progresses through generation of DNA variants followed by selection is a key learning outcome for biology students. We designed an integrated and innovative undergraduate laboratory exercise using Saccharomyces cerevisiae to demonstrate these principles. Students perform in vitro experimental evolution by repeatedly propagating large or small yeast colonies on a weekly basis. Small-colony variants known as petites arise by mutations that disrupt aerobic respiration. To demonstrate the effects of increased mutation rates, half of the selection lines are exposed to ultraviolet irradiation. To understand how the petite phenotype arises, polymerase chain reaction (PCR) is performed to examine mitochondrial DNA, while biochemical assays are used to assess the ability of petites to undergo aerobic respiration. This exercise demonstrates evolution by artificial selection over a suitably short timeframe and links the results to a critical biochemical process: the role of mitochondria in aerobic respiration and ATP production. By implementing these experiments, we successfully demonstrated that the frequencies of petite mutants in evolved populations varied according to the selection pressure we applied, and that petite mutants carried deletions in mitochondrial DNA as anticipated. Through an integrated learning context, this practical exercise promotes fundamental understanding of evolutionary processes and fosters critical thinking skills.

Saccharomyces cerevisiae is a useful eukaryotic model organism for demonstrating fundamental principles of various branches of biology (Botstein et al., 1997; Rinaldi, et al., 2010). In recent years, S. cerevisiae has been used as a versatile pedagogical resource for designing undergraduate laboratory courses that focus on experimental skills in molecular and evolutionary biology (Chan et al., 2021; Hageman & Krikken, 2018; Marshall, 2019; McDonnell et al., 2022; Ågren et al., 2017). We developed an integrated and highly customizable six- to seven-week practical exercise for undergraduates that capitalizes on the phenotypic plasticity and evolvability of S. cerevisiae.

Through this integrated learning context, we hope to promote critical thinking skills and encourage students to reflect on the ways in which various assays complement each other...

In both naturally evolving and mutagen-treated populations of S. cerevisiae, small-colony variants (SCVs) of S. cerevisiae frequently arise. They have slower growth rates and significantly smaller colony sizes compared with those of their ancestral strains (Garcia et al., 2019; Hess et al., 2009; Osman, et al., 2015). Petite mutants are SCVs that cannot undergo aerobic respiration and must rely on anaerobic respiration as their only source of ATP (Lipinski et al., 2010; Ogur et al., 1957; Powell et al., 2000). Cytoplasmic petites can be broadly classified as rho mutants that carry deleterious mutations in their mitochondrial DNA (mtDNA) and rho0 mutants that have completely lost their mtDNA. Nuclear petites (rho+) arise due to deleterious mutations in chromosomal genes that cause defects in oxidative phosphorylation. (Ferguson & von Borstel, 1992; Rinaldi et al., 2010) Due to the relevance of petites to fundamental questions in cell biology, research on S. cerevisiae petites has been carried out since the 1950s (Gibson et al., 2008; Ogur et al., 1957). Petites also have practical implications on the biomanufacturing industry. In the brewing industry for example, the accumulation of petite mutants in recycled yeast populations has negative impacts on fermentation, flocculation, and flavor components (Gibson et al., 2008; Lodolo et al., 2008).

Our experimental design was inspired by an integrated pedagogical approach that advocates providing learners an environment through which they can make connections between different concepts across disciplines (Basu et al., 2017; D’Souza et al., 2016). Using this laboratory exercise as a platform, we hope to provide undergraduates a more holistic learning experience by bridging the gap between key concepts in molecular, evolutionary, and cell biology, which are typically taught as independent academic modules at the undergraduate level.

The core component of this laboratory exercise involves students performing in vitro experimental evolution with the aim of isolating petite mutants. An ancestral population of S. cerevisiae BY4741 is repeatedly bottlenecked and passaged on yeast-peptone-dextrose (YPD) nutrient agar on a weekly basis (Brachmann et al., 1998). Variation in colony sizes allows experimental selection based on the two distinct colony sizes (small or large) to be applied. In half of the independently evolving lineages, only the smallest colonies are repeatedly passaged, which allowed the SCV phenotype to be selected for. In the other half of the lineages, only the largest colonies are repeatedly propagated to select against the SCV phenotype.

Exposing yeast to a range of mutagens favors the evolution of petite mutants (Ferguson & von Borstel, 1992; Goldring et al., 1971). To explore the effects of ultraviolet irradiation (UV) on the evolution of petites and the experimental outcomes, half of the experimental lineages in this exercise are irradiated with UV to increase mutation rate. Students receive training in PCR to detect cytoplasmic petites that have lost all or part of their mtDNA. Various phenotypic assays are performed to assess whether the evolved lineages are bona fide petites that are deficient in aerobic respiration. The exercise presented in this work adds to the growing literature of modern undergraduate-level experimental courses that focus on the roles of mutations in yeast (Marshall, 2019; McDonnell et al., 2022; Ågren et al., 2017). Through this integrated learning context, we hope to promote critical thinking skills and encourage students to reflect on the ways in which various assays complement each other in terms of linking phenotypic traits of petite mutants to their genotypes.

The learning objectives can be customized according to the curriculum requirements of the teaching institution. In the core component on experimental evolution, it is recommended that all students acquire technical competence in the following skill areas:

  • Effectively use aseptic techniques that are applicable across general microbiology to minimize microbial contamination.

  • Perform serial dilution on microbial cell suspensions in liquid nutrient medium.

  • Perform spread-plating on nutrient growth agar for cultivation of microbes.

  • Perform streak dilution of microbes on designated nutrient growth agars.

  • Collect and plot quantitative data on yeast colony numbers.

  • Perform PCR using standard molecular biology reagents and oligonucleotide primers.

  • Perform gel electrophoresis of PCR products on an agarose gel and interpret experimental results.

  • Understand the importance of including appropriate positive and negative control reactions when designing PCR experiments.

Upon successful completion of the exercise, students are expected to demonstrate conceptual understanding of the following topics at the interface between evolutionary, molecular, and basic cell biology:

  • Explain the concept of a population bottleneck and its roles in experimental evolution and natural selection.

  • Outline the main processes of UV-induced DNA damage and its effects on mutation rate.

  • Explain the concepts of deleterious mutations and compensatory adaptation.

  • Describe the role of glycolysis in producing pyruvate that is required for both aerobic and anaerobic respiration.

  • Understand the roles of mitochondria in generating energy via aerobic respiration.

  • Outline the role of oxidative phosphorylation in ATP production.

  • Explain why anaerobic respiration is less efficient than aerobic respiration in terms of energy production.

Experimental Evolution Assays

Students work in pairs throughout the experiments. Figure 1 provides an overview of the main procedures for the experimental evolution core component. Comprehensive protocols that are suitable for student use and an accompanying “Notes for Demonstrators” can be found in the Supplementary Materials provided with the online version of this article. During the Week 1 session, each pair of students pick a very small-colony and a very large-colony of S. cerevisiae BY4741 from a YPD nutrient agar plate for resuspension in YPD liquid medium and serial dilution according to the schematics in Figure 2 (Brachmann et al., 1998). Diluted cell suspensions of each colony size are spread plated onto YPD nutrient agar plates. Plates are incubated at room temperature for one week. In the Week 2 session, each colony size is passaged in duplicate sets on fresh YPD plates. One of the two sets for each colony size is irradiated with ultraviolet (UV) light of 254 nm wavelength for 25 seconds to increase mutation rate in these lineages.

Figure 1.

Overview of the procedures for experimental evolution of petite mutants of S. cerevisiae during the weekly teaching sessions in Weeks 1–4.

Figure 1.

Overview of the procedures for experimental evolution of petite mutants of S. cerevisiae during the weekly teaching sessions in Weeks 1–4.

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Figure 2.

Recommended procedures for the serial dilution of resuspended large or small colonies in YPD liquid medium prior to spread-plating on YPD agar plates. Abbreviations: S = small-colony lineages; L = large-colony lineages.

Figure 2.

Recommended procedures for the serial dilution of resuspended large or small colonies in YPD liquid medium prior to spread-plating on YPD agar plates. Abbreviations: S = small-colony lineages; L = large-colony lineages.

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In Weeks 3 and 4, the passaging procedure for the four independently evolving lineages corresponding to four different selection pressures is repeated. Each week, the number of SCVs are counted and calculated as a percentage of all the colonies for each of the four lineages. The frequencies of SCVs in the small-colony lineages are expected to increase over time. In contrast, the frequencies of SCVs are expected to decrease in the large-colony lineages, as SCVs in these lineages are repeatedly selected against. UV-irradiated lineages are expected to show higher rates of phenotypic evolution due to the activation of low-fidelity translesion synthesis (TLS) DNA polymerases (Guo et al., 2001; Lawrence & Christensen, 1976). In Week 5, quantitative data on the frequencies of SCVs across all available time points are pooled for a class-based data analysis exercise.

Phenotypic Characterization of Petite Mutants

To test whether the evolved SCVs are bona fide petites in terms of their deficiencies in aerobic respiration, resuspended endpoint colonies from each evolved lineage are streaked on yeast-peptone-glycerol (YPG) and yeast-peptone-ethanol (YPE) plates during the Week 5 teaching session. Petites cannot grow on media that only contain nonfermentable carbon sources such as glycerol and ethanol (Lipinski et al., 2010). During these procedures, students are shown how to perform streak dilution using inoculation loops.

A complementary phenotypic assay involves staining colonies using the 2,3,5-triphenyl tetrazolium chloride redox indicator. Yeast colonies that can perform aerobic respiration are stained dark red when a soft agar containing tetrazolium chloride is poured over the colonies. The color change is based on reduction of the tetrazolium salt by active mitochondrial dehydrogenases into formazan. Colonies that are deficient in aerobic respiration due to nuclear or mitochondrial mutations are unstained (Hess et al., 2009; Ogur et al., 1957). Using this technique, evolved endpoint colonies on YPD plates are screened for their ability to perform aerobic respiration during the teaching session in Week 5.

Most endpoint colonies from evolved small-colony lineages are expected to be bona fide petites that are unstained by tetrazolium chloride. The tetrazolium chloride overlay technique also distinguishes petite mutants from other non-petite SCVs, thus allowing petites to be more accurately identified. Detailed experimental protocols for all three phenotypic assays can be found in the “Notes for Demonstrators” in the Supplementary Materials provided with the online version of this article. The ancestral BY4741 strain should be included as a control strain in all the phenotypic assays.

Polymerase Chain Reaction

Students perform PCR to identify petites that have most likely lost their mtDNA. During the teaching session in Week 6, boiled lysates of glycerol stocks from yeast colonies on Weeks 2 and 4 YPD plates are used as templates for PCR-based detection of two mitochondrial genes (ATP9 and COX3) and a chromosomal gene ACT1 as positive control (Dirick et al., 2014; Osman et al., 2015).

The loss of ATP9 greatly increases mtDNA instability and the likelihood of mtDNA loss to form rho0 petite mutants (Bietenhader et al., 2012). Due to the possibility that some cytoplasmic petites could be rho mutants that have fixed large deletions in their mtDNA, it is essential to target at least two mitochondrial genes that are spaced far apart in mtDNA, such as ATP9 and COX3 (see Figure S4 in the Supplementary Materials provided with the online version of this article) (Osman et al., 2015). The oligonucleotide sequences of the three primers are summarized in Table S2 (see Supplementary Materials provided with the online version of this article). Boiled lysate of the ancestral BY4741 strain should be added to the positive control reactions as template, while negative control reactions without template should also be included.

Subsequently, all the PCR products are analyzed by gel electrophoresis. In all the sample groups, 100 bp bands corresponding to the chromosomal ACT1 gene fragment should be detected if alkaline lysis of yeast glycerol stocks was successfully carried out. For rho0 cytoplasmic petites without mtDNA, neither of the two mitochondrial genes will be amplified. Detection of only one out of the two tested mitochondrial genes suggests that the undetected gene was affected by deletion(s) in mtDNA. Demonstrators should emphasize that this PCR test has an inherent limitation in that it can identify neither rho+ nuclear petites nor rho cytoplasmic petites with mutations such as single nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs) that affect only a few nucleotides in mtDNA.

As a supplementary experiment to confirm that evolved ΔATP9 ΔCOX3 mutants are rho0 petites rather than rho petites with large deletions in their mtDNA, ΔATP9 ΔCOX3 petites can be treated with 4’,6-diamidino-2-phenylindole (DAPI) nuclear stain and imaged on a fluorescent microscope according to previously described experimental procedures (Amberg, et al., 2006; Dirick et al., 2014; Williamson & Fennell, 1979). Given the technical complexity of fluorescence microscopy procedures, we strongly recommend that this experiment should be performed by the demonstrators. Students can observe the processes of sample preparation, image requisition, and image analysis under guidance. As possible further extensions to this laboratory exercise, optional higher-level activities, such as Nanopore long-read sequencing of genomic DNA and mtDNA extracted from selected petite mutants, can be performed by technical staff.

This laboratory exercise was trialed and implemented by undergraduates in Biological Sciences under the laboratory guidance of a postdoctoral researcher at Macquarie University in 2022. The experimental evolution assay was performed in three independent lineages (n = 3) for each of the four selection pressures (large or small-colony lineages with or without UV-treatment), resulting in 12 evolved lineages in total. SCVs with distinctively small-colony sizes were observed since Week 1 of the experiment (Figure 3A), which allowed small-colony variant lineages to be established at the start of the experiment as planned.

Figure 3.

Phenotypes of S. cerevisiae BY4741 colonies on YPD agar plates. (A) SCVs with distinctively small-colony sizes were observed from Week 1 of the experiment, an example of which is circled in black. (B) Almost all the colonies of the S. cerevisiae BY4741 ancestral strain were stained dark red by the tetrazolium chloride overlay method, which demonstrates their ability to undergo aerobic respiration. (C) Similarly, most endpoint colonies of a representative large-colony lineage (L2) were stained dark red by tetrazolium chloride. (D) The endpoint colonies of a representative small-colony lineage (S2) showed the characteristic SCV phenotype and were mostly unstained by tetrazolium chloride.

Figure 3.

Phenotypes of S. cerevisiae BY4741 colonies on YPD agar plates. (A) SCVs with distinctively small-colony sizes were observed from Week 1 of the experiment, an example of which is circled in black. (B) Almost all the colonies of the S. cerevisiae BY4741 ancestral strain were stained dark red by the tetrazolium chloride overlay method, which demonstrates their ability to undergo aerobic respiration. (C) Similarly, most endpoint colonies of a representative large-colony lineage (L2) were stained dark red by tetrazolium chloride. (D) The endpoint colonies of a representative small-colony lineage (S2) showed the characteristic SCV phenotype and were mostly unstained by tetrazolium chloride.

Close modal

None of the endpoint populations from the small-colony lineages were able to grow on YPG agar and YPE agar plates, which suggests that yeast cells in those lineages had lost their ability to undergo aerobic respiration. We then performed tetrazolium chloride staining of the endpoint populations. Almost all the colonies of the S. cerevisiae BY4741 founding strain and the endpoint colonies of a representative large-colony lineage (L2) stained dark red (Figures 3B and 3C). In contrast, the endpoint colonies in a representative small-colony lineage (S2) were SCVs that were mostly unstained by tetrazolium chloride (Figure 3D).

In Weeks 3, 4, and 5, the frequencies of SCVs on YPD plates from the previous week were quantified and calculated as percentages of all the counted colonies on the same plates for each of the four selection pressures. The average frequency of SCVs in the small-colony lineages (n = 3) increased over time, which is consistent with the principle that sustained artificial selection leads to fixation of the selected trait (Figure 4A). The frequencies of SCVs decreased in the large-colony lineages (n = 3) over time because the SCV phenotype was repeatedly selected against (Figure 4B). By the end of the experiment, the average frequency of SCVs in the UV-irradiated small-colony lineages (n = 3) were significantly higher than that in the non-irradiated small-colony lineages (unpaired two-sample t-test: t = 3.31, d.f. = 4, p = .0297). This is consistent with our expectation that the rate of phenotypic evolution increases with higher mutation rate in the UV-irradiated lineages. The continued presence of non-SCV colonies in the small-colony lineages that are deficient in aerobic respiration could potentially be due to compensatory adaptation that ameliorates the fitness defects of petites (Garcia et al., 2019).

Figure 4.

Average frequencies (n = 3) of SCVs expressed as percentages of all counted colonies in (A) small-colony lineages with and without UV-treatment and in (B) large-colony lineages with and without UV-treatment. The lines representing UV-irradiated lineages are shown as dotted lines. The average frequency of SCVs in the UV-irradiated small-colony lineages were significantly higher than that in the non-irradiated small-colony lineages (unpaired two-sample t-test: t = 3.31, d.f. = 4, p = .0297).

Figure 4.

Average frequencies (n = 3) of SCVs expressed as percentages of all counted colonies in (A) small-colony lineages with and without UV-treatment and in (B) large-colony lineages with and without UV-treatment. The lines representing UV-irradiated lineages are shown as dotted lines. The average frequency of SCVs in the UV-irradiated small-colony lineages were significantly higher than that in the non-irradiated small-colony lineages (unpaired two-sample t-test: t = 3.31, d.f. = 4, p = .0297).

Close modal

Our PCR results showed that loss of mtDNA had most probably occurred in four out of six small-colony lineages (Table 1) because neither COX3 nor ATP9 can be detected in these lineages. Interestingly, only COX3, but not ATP9, was detected in the remaining two UV-irradiated small-colony lineages. This was most likely due to deletion mutations that resulted in the loss of ATP9, but not COX3, thus resulting in rho cytoplasmic petites. In contrast, all the large-colony lineages retained their mtDNA as expected. In terms of the general trend for all the three pairs of primers we tested, the PCR results for Week 4 colonies were identical to those from Week 2 (Table 1). In the positive control reactions, the chromosomal ACT1 gene was detected in all the samples with the exception of the no-template negative controls. This suggests that alkaline lysis of yeast cells and PCR were both correctly performed. Taken together, these results corroborate our phenotypic results for the evolved lineages and showed that parallel evolution can be observed in the three independent lineages for the four selection pressures.

Table 1.

PCR results for small- and large-colony evolved lineages. “+” in the table indicates detection of the respective PCR products of the correct band sizes. The “-” sign indicates their absence.

WeekmtDNA or chromosomal DNA targetGeneSmall-colony lineagesLarge-colony lineages
No UVWith UVNo UVWith UV
S1S2S3S1_UVS2_UVS3_UVL1L2L3L1_UVL2_UVL3_UV
mtDNA COX3 − − − − 
ATP9 − − − − − − 
Chromosomal ACT1 
mtDNA COX3 − − − − 
ATP9 − − − − − − 
Chromosomal ACT1 
WeekmtDNA or chromosomal DNA targetGeneSmall-colony lineagesLarge-colony lineages
No UVWith UVNo UVWith UV
S1S2S3S1_UVS2_UVS3_UVL1L2L3L1_UVL2_UVL3_UV
mtDNA COX3 − − − − 
ATP9 − − − − − − 
Chromosomal ACT1 
mtDNA COX3 − − − − 
ATP9 − − − − − − 
Chromosomal ACT1 

To help demonstrators assess whether students have acquired the theoretical concepts that underpin this laboratory exercise, we compiled two sets of assignment questions that focus on laboratory general knowledge—molecular and evolutionary biology—basic aspects of cellular microbiology (see Supplementary Materials provided with the online version of this article). A standardized set of grading criteria and sample answers are available upon request.

Detailed safety assessments must be performed prior to the start of the practical class in accordance with the health and safety regulations of the teaching institutions. All students require hands-on training from demonstrators on the safe use of Bunsen burners to create an aseptic working environment while completing microbiological procedures. Tetrazolium chloride is a chemical irritant that can cause skin and eye damage or corrosion. It may also cause respiratory irritation if accidentally inhaled. Therefore, it is recommended that demonstrators should prepare tetrazolium chloride stock solutions from powder in designated chemical hoods in advance. During the gel electrophoresis experiment, students need to wear disposable safety gloves when working with pre-stained agarose gels that contain any DNA-binding dye.

We sincerely thank Dr. Thomas Williams for the S. cerevisiae BY4741 strain and Mr. Mark Tran for loaning us the Vilber-Lourmat UV darkroom. We are also very grateful to the laboratory of Professor Ian Paulsen for the loan of tetrazolium chloride. Michael R Gillings and Sasha G Tetu are funded by the Australian Research Council Centre of Excellence in Synthetic Biology (CE200100029).

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Supplementary data