1887

Laboratory Exercise To Measure Restriction Enzyme Kinetics

    Authors: Caroline Blassick1, Benjamin David1, Audra Storm1,2, Paul Jensen1,2, Karin Jensen1,*
    VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801; 2: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Received 23 October 2018 Accepted 18 June 2019 Published 31 October 2019
    • ©2019 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 Bioengineering, University of Illinois at Urbana-Champaign, 1406 W. Green St., Urbana, IL 61801. Phone: 217-265-6941. E-mail: [email protected].
    Source: J. Microbiol. Biol. Educ. October 2019 vol. 20 no. 3 doi:10.1128/jmbe.v20i3.1703
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
  • XML
    24.84 Kb
  • PDF
    400.36 Kb
  • HTML
    27.25 Kb

    Abstract:

    Enzymes are ubiquitous in the fields of biology and microbiology, catalyzing critical reactions and enabling a broad range of biotechnological applications. Despite the important role that enzyme catalysis plays in biological processes, undergraduate students often struggle to understand enzyme kinetics in the classroom. In an attempt to improve students’ understanding of the topic, we present a relatively short and inexpensive laboratory activity designed to give students hands-on experience with generating and manipulating enzyme kinetic data. Students perform restriction digests of DNA at various time points, visualize the reaction products on an agarose gel, and quantify their data in order to construct Lineweaver-Burk plots which compare the effects of a restriction enzyme and its engineered version. The activity may be completed in a single two-hour lab session and, unlike other enzyme assays designed for laboratory courses, does not require a microplate reader to complete. The activity allows students to see connections between a visual data set and quantitative kinetic data, in order to solidify their understanding of enzyme kinetics. Students also learn the skills of gel electrophoresis and image quantification using ImageJ software. This lab activity is ideal for undergraduate laboratory courses which address enzyme kinetics and DNA technology.

References & Citations

1. Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH 2005 Evolving strategies for enzyme engineering Curr Opin Struct Biol 15 4 447 452 10.1016/j.sbi.2005.06.004 16006119 http://dx.doi.org/10.1016/j.sbi.2005.06.004
2. Linenberger KJ, Bretz SL 2015 Biochemistry students’ ideas about how an enzyme interacts with a substrate Biochem Mol Biol Educ 43 4 213 222 10.1002/bmb.20868 25850382 http://dx.doi.org/10.1002/bmb.20868
3. Loenen WA, Dryden DT, Raleigh EA, Wilson GG, Murray NE 2014 Highlights of the DNA cutters: a short history of the restriction enzymes Nucleic Acids Res 42 1 3 19 10.1093/nar/gkt990 http://dx.doi.org/10.1093/nar/gkt990
4. Flurkey WH, Inlow JK 2017 Use of mushroom tyrosinase to introduce Michaelis-Menten enzyme kinetics to biochemistry students Biochem Mol Biol Educ 45 3 270 276 10.1002/bmb.21029 28509370 http://dx.doi.org/10.1002/bmb.21029
5. Powers JL, Kiesman NE, Tran CM, Brown JH, Bevilacqua VL 2007 Lactate dehydrogenase kinetics and inhibition using a microplate reader Biochem Mol Biol Educ 35 4 287 292 10.1002/bmb.74 21591107 http://dx.doi.org/10.1002/bmb.74
6. Barton JS 2011 A comprehensive enzyme kinetic exercise for biochemistry J Chem Educ 88 9 1336 1339 10.1021/ed100816r http://dx.doi.org/10.1021/ed100816r
7. Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC 1968 Enzymatic breakage and joining of deoxyribonucleic acid VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4 J Biol Chem 243 17 4543 4555 4879167
8. Jin J, Vaud S, Zhelkovsky AM, Posfai J, McReynolds LA 2016 Sensitive and specific miRNA detection method using SplintR Ligase Nucleic Acids Res 44 13 e116 10.1093/nar/gkw399 27154271 5291259 http://dx.doi.org/10.1093/nar/gkw399
9. Kettling U, Koltermann A, Schwille P, Eigen M 1998 Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy Proc Natl Acad Sci U S A 95 4 1416 1420 10.1073/pnas.95.4.1416 9465029 19026 http://dx.doi.org/10.1073/pnas.95.4.1416
10. Schneider CA, Rasband WS, Eliceiri KW 2012 NIH Image to ImageJ: 25 years of image analysis Nat Methods 9 7 671 10.1038/nmeth.2089 22930834 5554542 http://dx.doi.org/10.1038/nmeth.2089

Supplemental Material

Loading

Article metrics loading...

/content/journal/jmbe/10.1128/jmbe.v20i3.1703
2019-10-31
2019-12-12

Abstract:

Enzymes are ubiquitous in the fields of biology and microbiology, catalyzing critical reactions and enabling a broad range of biotechnological applications. Despite the important role that enzyme catalysis plays in biological processes, undergraduate students often struggle to understand enzyme kinetics in the classroom. In an attempt to improve students’ understanding of the topic, we present a relatively short and inexpensive laboratory activity designed to give students hands-on experience with generating and manipulating enzyme kinetic data. Students perform restriction digests of DNA at various time points, visualize the reaction products on an agarose gel, and quantify their data in order to construct Lineweaver-Burk plots which compare the effects of a restriction enzyme and its engineered version. The activity may be completed in a single two-hour lab session and, unlike other enzyme assays designed for laboratory courses, does not require a microplate reader to complete. The activity allows students to see connections between a visual data set and quantitative kinetic data, in order to solidify their understanding of enzyme kinetics. Students also learn the skills of gel electrophoresis and image quantification using ImageJ software. This lab activity is ideal for undergraduate laboratory courses which address enzyme kinetics and DNA technology.

Highlighted Text: Show | Hide
Loading full text...

Full text loading...

/deliver/fulltext/jmbe/20/3/jmbe-20-53.html?itemId=/content/journal/jmbe/10.1128/jmbe.v20i3.1703&mimeType=html&fmt=ahah

Figures

Image of FIGURE 1

Click to view

FIGURE 1

DNA gel electrophoresis of BsaI and BsaI-HFv2 time course.

Source: J. Microbiol. Biol. Educ. October 2019 vol. 20 no. 3 doi:10.1128/jmbe.v20i3.1703
Download as Powerpoint
Image of FIGURE 2

Click to view

FIGURE 2

Best-fit velocity vs. substrate plots of BsaI and BsaI-HFv2 enzymes.

Source: J. Microbiol. Biol. Educ. October 2019 vol. 20 no. 3 doi:10.1128/jmbe.v20i3.1703
Download as Powerpoint
Image of FIGURE 3

Click to view

FIGURE 3

DNA gel electrophoresis of optional exercise to test BsaI-HFv2 for star activity.

Source: J. Microbiol. Biol. Educ. October 2019 vol. 20 no. 3 doi:10.1128/jmbe.v20i3.1703
Download as Powerpoint

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error