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 A 1972 Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3′ leads to 5′ exonuclease activity in deoxyribonucleic acid polymerases J Biol Chem 247 241 248 4336040
3. Frank EG, Woodgate R 2007 Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese J Biol Chem 282 24689 24696 10.1074/jbc.M702159200 17609217 http://dx.doi.org/10.1074/jbc.M702159200
4. Fujii R 2004 One-step random mutagenesis by error-prone rolling circle amplification Nucleic Acids Res 32 e145 10.1093/nar/gnh147 15507684 528823 http://dx.doi.org/10.1093/nar/gnh147
5. Labrou NE 2010 Random mutagenesis methods for in vitro directed enzyme evolution Curr Protein Pept Sci 11 91 100 10.2174/138920310790274617 20201809 http://dx.doi.org/10.2174/138920310790274617
6. Lai Y-P, Huang J, Wang L-F, Li J, Wu ZR 2004 A new approach to random mutagenesis in vitro Biotechnol Bioeng 86 622 627 10.1002/bit.20066 15137072 http://dx.doi.org/10.1002/bit.20066
7. Selifonova O, Valle F, Schellenberger V 2001 Rapid evolution of novel traits in microorganisms Appl Environ Microbiol 67 3645 3649 10.1128/AEM.67.8.3645-3649.2001 11472942 93066 http://dx.doi.org/10.1128/AEM.67.8.3645-3649.2001
8. Fujii R, Kitaoka M, Hayashi K 2014 Error-prone rolling circle amplification greatly simplifies random mutagenesis Methods Mol Biol 1179 23 29 10.1007/978-1-4939-1053-3_2 25055768 http://dx.doi.org/10.1007/978-1-4939-1053-3_2
9. Fujii R, Kitaoka M, Hayashi K 2006 Error-prone rolling circle amplification: the simplest random mutagenesis protocol Nat Protoc 1 2493 2497 10.1038/nprot.2006.403 http://dx.doi.org/10.1038/nprot.2006.403
10. Pierce BA 2013 Genetics: a conceptual approach 5th ed W. H. Freeman New York, NY
11. Zamft BM, Marblestone AH, Kording K, Schmidt D, Martin-Alarcon D, Tyo K, Boyden ES, Church G 2012 Measuring cation-dependent DNA polymerase fidelity landscapes by deep sequencing PLoS One 7 e43876 10.1371/journal.pone.0043876 http://dx.doi.org/10.1371/journal.pone.0043876
12. Lin-Goerke JL, Robbins DJ, Burczak JD 1997 PCR-based random mutagenesis using manganese and reduced dNTP concentration Biotechniques 23 409 412 9298207
13. Beckman RA, Mildvan AS, Loeb LA 1985 On the fidelity of DNA replication: manganese mutagenesis in vitro Biochemistry 24 5810 5817 10.1021/bi00342a019 3910084 http://dx.doi.org/10.1021/bi00342a019
14. Medin CL, Nolin KL A linked series of laboratory exercises in molecular biology utilizing bioinformatics and GFP Biochem Mol Biol Educ 39 448 456 22081550
15. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY 2004 Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein Nat Biotechnol 22 1567 1572 10.1038/nbt1037 15558047 http://dx.doi.org/10.1038/nbt1037
16. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY 2002 A monomeric red fluorescent protein Proc Natl Acad Sci U S A 99 7877 7882 10.1073/pnas.082243699 12060735 122988 http://dx.doi.org/10.1073/pnas.082243699
17. Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ 2006 Novel chromophores and buried charges control color in mFruits Biochemistry 45 9639 9647 10.1021/bi060773l 16893165 http://dx.doi.org/10.1021/bi060773l
18. de Boer HA, Comstock LJ, Vasser M 1983 The tac promoter: a functional hybrid derived from the trp and lac promoters Proc Natl Acad Sci U S A 80 21 25 10.1073/pnas.80.1.21 6337371 393301 http://dx.doi.org/10.1073/pnas.80.1.21
19. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular cloning: a laboratory manual Cold Spring Harbor Laboratory Cold Spring Harbor, NY
20. Ahn SC, Baek BS, Oh T, Song CS, Chatterjee B 2000 Rapid mini-scale plasmid isolation for DNA sequencing and restriction mapping Biotechniques 29 466 468 10997259

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2017-04-21
2019-07-16

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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