1887

Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

    Authors: Betsy M. Martinez-Vaz1,*, Irina Makarevitch1, Shane Stensland1
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    Affiliations: 1: Department of Biology, Hamline University, Saint Paul, MN 55104
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 20 May 2010
    • Supplemental material available at http://jmbe.asm.org
    • *Corresponding author. Mailing address: Hamline University, Department of Biology, 1536 Hewitt Ave., Saint Paul, MN 55104. Phone: (651) 523-2493. Fax: (651) 523-2620. E-mail: [email protected].
    • Copyright © 2010 American Society for Microbiology
    Source: J. Microbiol. Biol. Educ. May 2010 vol. 11 no. 1 42-49. doi:10.1128/jmbe.v11.i1.101
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    Abstract:

    A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation.

Key Concept Ranking

Gene Expression and Regulation
0.9304534
Transcription Factor Regulation
0.5406631
Gene Expression
0.4899491
Transcriptional Regulation
0.4609023
Gene Regulation
0.44810122
0.9304534

References & Citations

1. Blauwkamp TA, Ninfa AJ 2002 Nac-mediated repression of the serA promoter of Escherichia coli Mol. Microbiol. 45 351 363 10.1046/j.1365-2958.2002.02994.x 12123449 http://dx.doi.org/10.1046/j.1365-2958.2002.02994.x
2. Cormack BP, Valdivia R, Falkow S 1996 FACS-optimized mutants of the green fluorescent protein (GFP) Gene 173 33 38 10.1016/0378-1119(95)00685-0 8707053 http://dx.doi.org/10.1016/0378-1119(95)00685-0
3. Datsenko KA, Wanner BL 2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Proc Natl Acad Sci U S A 97 6640 6645 10.1073/pnas.120163297 10829079 http://dx.doi.org/10.1073/pnas.120163297
4. Ernsting BR, Denninger JW, Blumenthal RM, Matthews RG 1993 Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein? J. Bacteriol. 175 7160 7169 7901196
5. Keseler IM, Collado-Vides J, Gama-Castro S, et al 2005 EcoCyc: a comprehensive database resource for Escherichia coli Nucleic Acids Res. 33 Database issue D334 D337 10.1093/nar/gki108 540062 http://dx.doi.org/10.1093/nar/gki108
6. Mosher R 2002 Using pGLO to demonstrate the effects of catabolite repression on gene expression in Escherichia coli [abstract] Bioscene 28 17 23
7. Moss R 1997 A discovery lab for studying gene regulation The American Biology Teacher 59 522 526 10.2307/4450370 http://dx.doi.org/10.2307/4450370
8. National Research Council 2003 Bio2010: Transforming Undergraduate Education for Future Research Biologists National Academies Press Washington, DC
9. Oliver G, Gosset G, Sanchez-Pescador R, et al 1987 Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12 Gene 60 1 11 10.1016/0378-1119(87)90207-1 3326786 http://dx.doi.org/10.1016/0378-1119(87)90207-1
10. Paul L, Mishra PK, Blumenthal RM, Matthews RG 2007 Integration of regulatory signals through involvement of multiple global regulators: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR BMC Microbiol. 7 2 10.1186/1471-2180-7-2 17233899 1784095 http://dx.doi.org/10.1186/1471-2180-7-2
11. Salgado H, Gama-Castro S, Peralta-Gil M, et al 2006 RegulonDB (version 5.0): Escherichia coli K-12 transcriptional regulatory network, operon organization, and growth conditions Nucleic Acids Res. 34 Database issue D394 D397 10.1093/nar/gkj156 1347518 http://dx.doi.org/10.1093/nar/gkj156
12. Sambrook J, Russell DW 2001 Molecular Cloning: A Laboratory Manual Third Edition CHSL Press Cold Spring Harbor, NY
13. Stambuck B 2002 Transcriptional and posttranslational regulation of a membrane nutrient transporter Biochemistry and Molecular Biology Education 30 388 393 10.1002/bmb.2002.494030060136 http://dx.doi.org/10.1002/bmb.2002.494030060136
14. Tobey KL, Grant GA 1986 The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase J. Biol. Chem. 261 12179 12183 3017965
15. Wu Y, Zhou Y, Song J, Hu X, Ding Y, Zhang Z 2008 Using green and red fluorescent proteins to teach protein expression, purification, and crystallization Biochemistry and Molecular Biology Education 36 43 54 10.1002/bmb.117 21591159 http://dx.doi.org/10.1002/bmb.117
16. Zaslaver A, Mayo AE, Rosenberg R, et al 2004 Just-in-time transcription program in metabolic pathways Nat Genet 36 486 491 10.1038/ng1348 15107854 http://dx.doi.org/10.1038/ng1348

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2010-05-20
2019-01-16

Abstract:

A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation.

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Figures

Image of FIGURE 1

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FIGURE 1

Experimental data showing the activity of the promoter in cells grown on rich and minimal media. The data shown is an average of six replicas. A paired t-test was performed to evaluate the significance of the results; the data were significant and had a value of less than 0.000002. Fluorescence measurements were obtained after cultures were grown for two hours and had reached an OD600 of 0.4.

Source: J. Microbiol. Biol. Educ. May 2010 vol. 11 no. 1 42-49. doi:10.1128/jmbe.v11.i1.101
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Image of FIGURE 2

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FIGURE 2

An Example of experimental data obtained by the students. The activity of and promoters was studied in wild type cells and cells lacking L, a transcriptional regulator of both genes. Cells were grown on minimal medium to observe optimal promoter activity for and . The student’s data confirmed that L is a transcriptional activator of and . The data shown is the average of three replicas; error bars indicate the standard error of the mean values.

Source: J. Microbiol. Biol. Educ. May 2010 vol. 11 no. 1 42-49. doi:10.1128/jmbe.v11.i1.101
Download as Powerpoint

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