1887

Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation

    Authors: Jessica Cole1, Amanda Ferguson2, Verónica A. Segarra3, Susan Walsh2,*
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    Affiliations: 1: Department of Biology, Portland State University, Portland, OR 97207-0751; 2: Department of Biology, Rollins College, Winter Park, FL 32789; 3: Department of Biology, High Point University, High Point, NC 27268
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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    Abstract:

    Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions.

Key Concept Ranking

Bacterial Proteins
0.68060654
Point Mutation
0.5956499
Lac Repressor
0.51111114
0.68060654

References & Citations

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2. Brutlag D, Kornberg A1972Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3′ leads to 5′ exonuclease activity in deoxyribonucleic acid polymerasesJ Biol Chem2472412484336040
3. Frank EG, Woodgate R2007Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganeseJ Biol Chem282246892469610.1074/jbc.M70215920017609217 http://dx.doi.org/10.1074/jbc.M702159200
4. Fujii R2004One-step random mutagenesis by error-prone rolling circle amplificationNucleic Acids Res32e14510.1093/nar/gnh14715507684528823 http://dx.doi.org/10.1093/nar/gnh147
5. Labrou NE2010Random mutagenesis methods for in vitro directed enzyme evolutionCurr Protein Pept Sci119110010.2174/13892031079027461720201809 http://dx.doi.org/10.2174/138920310790274617
6. Lai Y-P, Huang J, Wang L-F, Li J, Wu ZR2004A new approach to random mutagenesis in vitroBiotechnol Bioeng8662262710.1002/bit.2006615137072 http://dx.doi.org/10.1002/bit.20066
7. Selifonova O, Valle F, Schellenberger V2001Rapid evolution of novel traits in microorganismsAppl Environ Microbiol673645364910.1128/AEM.67.8.3645-3649.20011147294293066 http://dx.doi.org/10.1128/AEM.67.8.3645-3649.2001
8. Fujii R, Kitaoka M, Hayashi K2014Error-prone rolling circle amplification greatly simplifies random mutagenesisMethods Mol Biol1179232910.1007/978-1-4939-1053-3_225055768 http://dx.doi.org/10.1007/978-1-4939-1053-3_2
9. Fujii R, Kitaoka M, Hayashi K2006Error-prone rolling circle amplification: the simplest random mutagenesis protocolNat Protoc12493249710.1038/nprot.2006.403 http://dx.doi.org/10.1038/nprot.2006.403
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11. Zamft BM, Marblestone AH, Kording K, Schmidt D, Martin-Alarcon D, Tyo K, Boyden ES, Church G2012Measuring cation-dependent DNA polymerase fidelity landscapes by deep sequencingPLoS One7e4387610.1371/journal.pone.0043876 http://dx.doi.org/10.1371/journal.pone.0043876
12. Lin-Goerke JL, Robbins DJ, Burczak JD1997PCR-based random mutagenesis using manganese and reduced dNTP concentrationBiotechniques234094129298207
13. Beckman RA, Mildvan AS, Loeb LA1985On the fidelity of DNA replication: manganese mutagenesis in vitroBiochemistry245810581710.1021/bi00342a0193910084 http://dx.doi.org/10.1021/bi00342a019
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15. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY2004Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteinNat Biotechnol221567157210.1038/nbt103715558047 http://dx.doi.org/10.1038/nbt1037
16. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY2002A monomeric red fluorescent proteinProc Natl Acad Sci U S A997877788210.1073/pnas.08224369912060735122988 http://dx.doi.org/10.1073/pnas.082243699
17. Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ2006Novel chromophores and buried charges control color in mFruitsBiochemistry459639964710.1021/bi060773l16893165 http://dx.doi.org/10.1021/bi060773l
18. de Boer HA, Comstock LJ, Vasser M1983The tac promoter: a functional hybrid derived from the trp and lac promotersProc Natl Acad Sci U S A80212510.1073/pnas.80.1.216337371393301 http://dx.doi.org/10.1073/pnas.80.1.21
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2017-04-21
2017-07-21

Abstract:

Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions.

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Figures

Image of FIGURE 1

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FIGURE 1

A schematic outlining the experimental procedures. X’s indicate mutations generated through RCA. In the sequence analysis at left, the mutant has a missense mutation (G5D). RCA = rolling circle amplification.

Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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Image of FIGURE 2

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FIGURE 2

The percentage of pink colonies decreases with increasing MnCl. Compiled student colony counts from the transformation in one course section are represented with varying treatments of MnCl. Because student pairs could select different experimental conditions, sample sizes vary. Standard deviation is indicated as error bars. For the following concentrations: 0 mM, 6; 0.25 mM, 1; 0.5 mM, 3; 1 mM, 4; and 1.5 mM, 3. RCA = rolling circle amplification.

Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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Image of FIGURE 3

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FIGURE 3

Colonies display phenotypic differences, specifically a higher percentage of white colonies as more manganese chloride is added. (A) plated on LB with ampicillin from transformations of RCA reactions. Left to right: negative control, 0.25 mM MnCl, 0.5 mM MnCl. (B) Brightfield microscopy of three colonies on a plate. (C) Fluorescent microscopy of the same colonies as in B. RCA = rolling circle amplification.

Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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Image of FIGURE 4

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FIGURE 4

A colony producing no color also produces no protein after induction with IPTG. (A) Control, white, and dark pink cultures are shown after induction with 1 mM IPTG. (B) Protein samples from the cultures in A were resolved by SDS-PAGE and stained with Coomassie blue. U is uninduced before IPTG, and I is induced. IPTG = isopropyl beta-D-thiogalactopyranoside.

Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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Image of FIGURE 5

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FIGURE 5

Cherry coding region sequences of nine white colonies from the same RCA show variation in mutations. Mutations are summarized as follows: 460: missense K173E; 468: missense G138D; 467: missense G25D; 459: frameshift at 139; 470: missense G57D and frameshift at 94; 464: missense I12F and frameshift at 118.Three sequences are not shown since they did not have mutations in the Cherry sequence; these data do not rule out mutations in the GST coding region or the promoter. RCA = rolling circle amplification. GST = glutathione transferase.

Source: J. Microbiol. Biol. Educ. April 2017 vol. 18 no. 1 doi:10.1128/jmbe.v18i1.1201
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