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Metagenomic Approaches to Identify Novel Organisms from the Soil Environment in a Classroom Setting

    Authors: Sadia J. Rahman1, Trevor C. Charles2, Parjit Kaur1,*
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    Affiliations: 1: Department of Biology, Georgia State University, Atlanta, GA, 30303, USA; 2: Department of Biology, University of Waterloo, Waterloo, ON N2V 2P1, Canada
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 02 December 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/ and 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 author. Mailing address: Department of Biology, Georgia State University, 58 Edgewood Ave., Atlanta, GA 30303. Phone: 404-413-5405. Fax: 404-413-5301. E-mail: [email protected].
    Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 423-429. doi:10.1128/jmbe.v17i3.1115
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    Abstract:

    Molecular Microbial Metagenomics is a research-based undergraduate course developed at Georgia State University. This semester-long course provides hands-on research experience in the area of microbial diversity and introduces molecular approaches to study diversity. Students are part of an ongoing research project that uses metagenomic approaches to isolate clones containing 16S ribosomal ribonucleic acid (rRNA) genes from a soil metagenomic library. These approaches not only provide a measure of microbial diversity in the sample but may also allow discovery of novel organisms. Metagenomic approaches differ from the traditional culturing methods in that they use molecular analysis of community deoxyribonucleic acid (DNA) instead of culturing individual organisms. Groups of students select a batch of 100 clones from a metagenomic library. Using universal primers to amplify 16S rRNA genes from the pool of DNA isolated from 100 clones, and a stepwise process of elimination, each group isolates individual clones containing 16S rRNA genes within their batch of 100 clones. The amplified 16S rRNA genes are sequenced and analyzed using bioinformatics tools to determine whether the rRNA gene belongs to a novel organism. This course provides avenues for active learning and enhances students’ conceptual understanding of microbial diversity. Average scores on six assessment methods used during field testing indicated that success in achieving different learning objectives varied between 84% and 95%, with 65% of the students demonstrating complete grasp of the project based on the end-of-project lab report. The authentic research experience obtained in this course is also expected to result in more undergraduates choosing research-based graduate programs or careers.

Key Concept Ranking

Bacteria and Archaea
0.59849423
16s rRNA Sequencing
0.51974165
0.59849423

References & Citations

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2. American Association for the Advancement of Science 2011 Vision and change in undergraduate biology education: a call to action: a summary of recommendations made at a national conference organized by the American Association for the Advancement of Science July 15–17, 2009 Washington, DC
3. Beja O, et al 2000 Bacterial rhodopsin: evidence for a new type of phototrophy in the sea Science 289 5486 1902 1906 10.1126/science.289.5486.1902 10988064 http://dx.doi.org/10.1126/science.289.5486.1902
4. Bintrim SB, Donohue TJ, Handelsman J, Roberts GP, Goodman RM 1997 Molecular phylogeny of Archaea from soil Proc Natl Acad Sci USA 94 1 277 282 10.1073/pnas.94.1.277 8990199 19314 http://dx.doi.org/10.1073/pnas.94.1.277
5. Donato JJ, Kllimstra MA, Byrnes JR, White RJ, Marsh TC 2012 The introduction of metagenomics into an undergraduate biochemistry laboratory course yielded a predicted reductase that decreases triclosan susceptibility in Escherichia coli DNA Cell Bio 31 6 968 973 10.1089/dna.2011.1512 http://dx.doi.org/10.1089/dna.2011.1512
6. Fuhrman JA 2012 Metagenomics and its connection to microbial community organization F1000 Biol Rep 4 15 10.3410/B4-15 22912649 3410722 http://dx.doi.org/10.3410/B4-15
7. Gasper BJ, Gardner SM 2013 Engaging students in authentic microbiology research in an introductory biology laboratory course is correlated with gains in student understanding of the nature of authentic research and critical thinking J Microbiol Biol Educ 14 1 25 34 10.1128/jmbe.v14i1.460 23858351 3706163 http://dx.doi.org/10.1128/jmbe.v14i1.460
8. Gibbens BB, Scott CL, Hoff CD, Schottel JL 2015 Exploring metagenomics in the laboratory of an introductory biology course J Microbiol Biol Educ 16 1 34 40 10.1128/jmbe.v16i1.780 25949755 4416502 http://dx.doi.org/10.1128/jmbe.v16i1.780
9. Handelsman J 2004 Metagenomics: application of genomics to uncultured microorganisms Microbiol Mol Biol Rev 68 4 669 685 10.1128/MMBR.68.4.669-685.2004 15590779 539003 http://dx.doi.org/10.1128/MMBR.68.4.669-685.2004
10. Hugenholtz P, Pace NR 1996 Identifying microbial diversity in the natural environment: a molecular phylogenetic approach Trends Biotechnol 14 6 190 197 10.1016/0167-7799(96)10025-1 8663938 http://dx.doi.org/10.1016/0167-7799(96)10025-1
11. Kaur P, Rahman SJ 2015 Molecular microbial metagenomics BIOL 4905 laboratory manual University Readers, Georgia State University Atlanta, GA
12. Muth TR, McEntee CM 2014 Undergraduate urban metagenomics research module J Microbiol Biol Educ 15 1 38 40 10.1128/jmbe.v15i1.645 24839517 4004741 http://dx.doi.org/10.1128/jmbe.v15i1.645
13. Neufeld JD, et al 2011 Open resource metagenomics: a model for sharing metagenomic libraries Stand Genomic Sci 5 2 203 210 10.4056/sigs.1974654 22180823 3235511 http://dx.doi.org/10.4056/sigs.1974654
14. Pace NR 1997 A molecular view of microbial diversity and the biosphere Science 276 5313 734 740 10.1126/science.276.5313.734 9115194 http://dx.doi.org/10.1126/science.276.5313.734
15. Rios-Velazquez C, et al 2011 Summer workshop in metagenomics: one week plus eight students equals gigabases of cloned DNA J Microbiol Biol Educ 12 2 120 126 10.1128/jmbe.v12i2.177 23653755 3577266 http://dx.doi.org/10.1128/jmbe.v12i2.177
16. Russell SH, Hancock MP, McCullough J 2007 Benefits of undergraduate research experience Sci Educ Forum 316 548 549
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2016-12-02
2019-05-26

Abstract:

Molecular Microbial Metagenomics is a research-based undergraduate course developed at Georgia State University. This semester-long course provides hands-on research experience in the area of microbial diversity and introduces molecular approaches to study diversity. Students are part of an ongoing research project that uses metagenomic approaches to isolate clones containing 16S ribosomal ribonucleic acid (rRNA) genes from a soil metagenomic library. These approaches not only provide a measure of microbial diversity in the sample but may also allow discovery of novel organisms. Metagenomic approaches differ from the traditional culturing methods in that they use molecular analysis of community deoxyribonucleic acid (DNA) instead of culturing individual organisms. Groups of students select a batch of 100 clones from a metagenomic library. Using universal primers to amplify 16S rRNA genes from the pool of DNA isolated from 100 clones, and a stepwise process of elimination, each group isolates individual clones containing 16S rRNA genes within their batch of 100 clones. The amplified 16S rRNA genes are sequenced and analyzed using bioinformatics tools to determine whether the rRNA gene belongs to a novel organism. This course provides avenues for active learning and enhances students’ conceptual understanding of microbial diversity. Average scores on six assessment methods used during field testing indicated that success in achieving different learning objectives varied between 84% and 95%, with 65% of the students demonstrating complete grasp of the project based on the end-of-project lab report. The authentic research experience obtained in this course is also expected to result in more undergraduates choosing research-based graduate programs or careers.

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Figures

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FIGURE 1

Flow of experiments in the project and analyses of PCR-amplified DNA. (A) Progression of experiments. A simplified flowchart of the experiments for identifying 16S rRNA gene-containing clones from the metagenomic library by a stepwise process of elimination. Red plus sign indicates the presence of a positive 16S-containing clone in the batch. (B) Isolation of pooled DNA and PCR analysis. This flowchart shows the experimental steps corresponding to each blue asterisk in panel A. DNA = deoxyribonucleic acid; rRNA = ribosomal ribonucleic acid; PCR = polymerase chain reaction; RDP = ribosomal database project.

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 423-429. doi:10.1128/jmbe.v17i3.1115
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FIGURE 2

Learning gains evident from pre/post-class surveys given during the Summer Pilot Program. A pre-class survey ( Appendix 3 ) was given on the first day of the program to test knowledge of the topics related to the course. The same survey was provided to the students at the end of the program. The surveys were completed by five students and were evaluated on accuracy as well as the extent of knowledge displayed. Blue = pre-class survey; red = post-class survey; RNA = ribonucleic acid; PCR = polymerase chain reaction; DNA = deoxyribonucleic acid.

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 423-429. doi:10.1128/jmbe.v17i3.1115
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FIGURE 3

Assessment of learning gains from two semester-long courses with a total of 16 students. (A) Average scores on all assessment methods ( 1 6 ) and the average overall course grade over two semesters ( 7 ). (B) Average scores on selected quiz questions (provided in Appendix 4.1 ). (C) Detailed assessment of student lab reports: writing style follows scientific paper (1); abstract summarizes purpose of the project (2); introduction includes appropriate content (3); method section provides concise narrative (4); results include rationale and summary of experiments (5); figures and tables are included in results (6); discussion includes analysis of results (7). (D) Overall assessment of the lab reports using a two-point rubric: i) understanding flow of the project, and ii) understanding central concepts. BLAST = basic local alignment search tool.

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 423-429. doi:10.1128/jmbe.v17i3.1115
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