Battle of the Bacteria: Characterizing the Evolutionary Advantage of Stationary Phase Growth †
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Authors:
Karin E. Kram1,*,
Kristina M. Yim2,
Aaron B. Coleman3,
Brian K. Sato4,*
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Published 04 May 2016
- ©2016 Author(s). Published by the American Society for Microbiology.
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[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
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Supplemental materials available at
http://asmscience.org/jmbe - *Corresponding authors. Mailing addresses: Brian Sato: 2238 McGaugh Hall MC3900, Irvine, CA 92697. Phone: 949-824-0661. Fax: 949-824-8551. E-mail: [email protected]; Karin Kram: 1000 E. Victoria St. NSM A-137, Carson, CA 90747. Phone: 310-243-1090. Fax: 301-243-2350. E-mail: [email protected].
Abstract:
Providing students with authentic research opportunities has been shown to enhance learning and increase retention in STEM majors. Accordingly, we have developed a novel microbiology lab module, which focuses on the molecular mechanisms of evolution in E. coli, by examining the growth advantage in stationary phase (GASP) phenotype. The GASP phenotype is demonstrated by growing cells into long-term stationary phase (LTSP) and then competing them against un-aged cells in a fresh culture. This module includes learning goals related to strengthening practical laboratory skills and improving student understanding of evolution. In addition, the students generate novel data regarding the effects of different environmental stresses on GASP and the relationship between evolution, genotypic change, mutation frequency, and cell stress. Pairs of students are provided with the experimental background, select a specific aspect of the growth medium to modify, and generate a hypothesis regarding how this alteration will impact the GASP phenotype. From this module, we have demonstrated that students are able to achieve the established learning goals and have produced data that has furthered our understanding of the GASP phenotype. Journal of Microbiology & Biology Education
References & Citations
Supplemental Material
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Appendix 1: Reagent/equipment list and faculty instructions
Appendix 2: Student handout and protocol
Appendix 3: Lecture slides
Appendix 4: GASP worksheet
Appendix 5: Module pre-/post-test
Appendix 6: Self-assessment and exam questions
Appendix 7: Pipetting test instructions
Appendix 8: Example student hypotheses
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Abstract:
Providing students with authentic research opportunities has been shown to enhance learning and increase retention in STEM majors. Accordingly, we have developed a novel microbiology lab module, which focuses on the molecular mechanisms of evolution in E. coli, by examining the growth advantage in stationary phase (GASP) phenotype. The GASP phenotype is demonstrated by growing cells into long-term stationary phase (LTSP) and then competing them against un-aged cells in a fresh culture. This module includes learning goals related to strengthening practical laboratory skills and improving student understanding of evolution. In addition, the students generate novel data regarding the effects of different environmental stresses on GASP and the relationship between evolution, genotypic change, mutation frequency, and cell stress. Pairs of students are provided with the experimental background, select a specific aspect of the growth medium to modify, and generate a hypothesis regarding how this alteration will impact the GASP phenotype. From this module, we have demonstrated that students are able to achieve the established learning goals and have produced data that has furthered our understanding of the GASP phenotype. Journal of Microbiology & Biology Education

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Author and Article Information
-
Published 04 May 2016
- ©2016 Author(s). Published by the American Society for Microbiology.
-
[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
-
Supplemental materials available at
http://asmscience.org/jmbe - *Corresponding authors. Mailing addresses: Brian Sato: 2238 McGaugh Hall MC3900, Irvine, CA 92697. Phone: 949-824-0661. Fax: 949-824-8551. E-mail: [email protected]; Karin Kram: 1000 E. Victoria St. NSM A-137, Carson, CA 90747. Phone: 310-243-1090. Fax: 301-243-2350. E-mail: [email protected].
Figures
Timeline for the four experiments in the GASP module. The module was implemented in a lab course that met twice a week (Lab Period A and B). Lab Period A was three hours in duration while Period B was one hour. Each activity during lab is associated with one of the four specific experiments. (1) Growth of cells into long-term stationary phase and GASP assay, (2) Examination of RpoS activity, (3) Cell stress measurement, (4) Examination of mutation frequency. Specific times required for each activity are indicated on the figure in minutes.

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FIGURE 1
Timeline for the four experiments in the GASP module. The module was implemented in a lab course that met twice a week (Lab Period A and B). Lab Period A was three hours in duration while Period B was one hour. Each activity during lab is associated with one of the four specific experiments. (1) Growth of cells into long-term stationary phase and GASP assay, (2) Examination of RpoS activity, (3) Cell stress measurement, (4) Examination of mutation frequency. Specific times required for each activity are indicated on the figure in minutes.
Examples of student data illustrating the GASP phenotype. (A) Dilution plating on selective media. Cells are from the co-culture experiment (started in week three of the module). Day 0 refers to the initial culture inoculation and day 7 refers to one week of co-culture growth. Aged cells are distinguished from un-aged cells by the presence of distinct antibiotic resistance markers present in each strain. Cultures are plated with ten-fold serial dilutions. (B) Colony forming units (CFU) per ml of culture are calculated from the dilution plates and graphed on a plot of CFU/ml versus time. The GASP phenotype is illustrated by greater survival values of aged cells over time compared to the un-aged population in the culture. Growth in different types of media can impact the GASP phenotype.

Click to view
FIGURE 2
Examples of student data illustrating the GASP phenotype. (A) Dilution plating on selective media. Cells are from the co-culture experiment (started in week three of the module). Day 0 refers to the initial culture inoculation and day 7 refers to one week of co-culture growth. Aged cells are distinguished from un-aged cells by the presence of distinct antibiotic resistance markers present in each strain. Cultures are plated with ten-fold serial dilutions. (B) Colony forming units (CFU) per ml of culture are calculated from the dilution plates and graphed on a plot of CFU/ml versus time. The GASP phenotype is illustrated by greater survival values of aged cells over time compared to the un-aged population in the culture. Growth in different types of media can impact the GASP phenotype.
Assessments confirm that students achieved the module learning objectives. (A) A 12-question pre-/post-test was administered before and after the GASP module in laboratory sections during Fall quarter 2014 and Winter quarter 2015 (n = 197 students combined). Performance on each question (Q) and the corresponding learning objective (LO) it assessed is indicated. All post-test gains are statistically significant (p < 0.001 by t-test) with the exception of question 4. (B) Students noted their agreement with the indicated statements on a 5 -point Likert scale (5 = strongly agree, 1 = strongly disagree) upon completion of the module. They were asked to state their current agreement and provide a retroactive agreement (the pre-module data). Post-test gains are statistically significant (p < 0.001 by chi-square test). Questions were asked using the iClicker response system (n = 190 students). (C) Student performance on the pipetting test before and after module completion (n = 198 students). Passing refers to whether a student’s dilution fell within 0.050 OD595 units from a standard value obtained by the course instructor who performed the same dilutions multiple times. The number of students who passed post-module was significantly greater than pre-module (p < 0.001 by t-test). (D) Student performance on exam questions categorized by whether or not they were GASP module-specific and by Bloom’s level (Bloom’s 2 versus 3, 4, and 5) (n = 201 students). The difference between GASP and Other questions of Bloom’s level 3, 4, and 5 was statistically significant (p < 0.001 by t-test). Question numbers in each category were GASP (BL2) = 5, Other (BL2) = 10, GASP (BL3, 4, 5) = 19, Other (BL3, 4, 5) = 38.

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FIGURE 3
Assessments confirm that students achieved the module learning objectives. (A) A 12-question pre-/post-test was administered before and after the GASP module in laboratory sections during Fall quarter 2014 and Winter quarter 2015 (n = 197 students combined). Performance on each question (Q) and the corresponding learning objective (LO) it assessed is indicated. All post-test gains are statistically significant (p < 0.001 by t-test) with the exception of question 4. (B) Students noted their agreement with the indicated statements on a 5 -point Likert scale (5 = strongly agree, 1 = strongly disagree) upon completion of the module. They were asked to state their current agreement and provide a retroactive agreement (the pre-module data). Post-test gains are statistically significant (p < 0.001 by chi-square test). Questions were asked using the iClicker response system (n = 190 students). (C) Student performance on the pipetting test before and after module completion (n = 198 students). Passing refers to whether a student’s dilution fell within 0.050 OD595 units from a standard value obtained by the course instructor who performed the same dilutions multiple times. The number of students who passed post-module was significantly greater than pre-module (p < 0.001 by t-test). (D) Student performance on exam questions categorized by whether or not they were GASP module-specific and by Bloom’s level (Bloom’s 2 versus 3, 4, and 5) (n = 201 students). The difference between GASP and Other questions of Bloom’s level 3, 4, and 5 was statistically significant (p < 0.001 by t-test). Question numbers in each category were GASP (BL2) = 5, Other (BL2) = 10, GASP (BL3, 4, 5) = 19, Other (BL3, 4, 5) = 38.