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Single-Step Gene Knockout of the Gene in : A Laboratory Exercise for Undergraduate Students

    Authors: Jurre Hageman1,*, Arjen M. Krikken2
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    Affiliations: 1: Expertise Centre ALIFE, Institute for Life Science & Technology, Hanze University of Applied Sciences, Groningen, Groningen, the Netherlands; 2: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    Source: J. Microbiol. Biol. Educ. October 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1615
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    Abstract:

    This article describes a relatively straightforward procedure to knock out the gene that encodes the invertase enzyme in baker’s yeast. The SUC2 gene, which encodes for the invertase enzyme, is knocked out by a single-step PCR knock out method. The knockout is subsequently confirmed at the genetic level by PCR and agarose gel electrophoresis. The knockout is confirmed at the biochemical level by measuring the activity of the invertase enzyme using a colorimetric assay. This tips and tools article describes an easily scalable, inexpensive, yet challenging research project helping undergraduate students at the Bachelor level to conceptualize the effect of the deletion of a gene encoding an enzyme.

References & Citations

1. Rothstein R1991Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeastMethods Enzymol19428130110.1016/0076-6879(91)94022-52005793 http://dx.doi.org/10.1016/0076-6879(91)94022-5
2. Wendland J2003PCR-based methods facilitate targeted gene manipulations and cloning proceduresCurr Genet4411512310.1007/s00294-003-0436-x12928752 http://dx.doi.org/10.1007/s00294-003-0436-x
3. Hinnebusch AG, Johnston M2011YeastBook: an encyclopedia of the reference eukaryotic cellGenetics18968368410.1534/genetics.111.135129220844193213385 http://dx.doi.org/10.1534/genetics.111.135129
4. Myrback K1957Studies on yeast invertase; soluble and insoluble invertase (saccharase) of baker’s yeastArch Biochem Biophys6913814810.1016/0003-9861(57)90481-213445188 http://dx.doi.org/10.1016/0003-9861(57)90481-2
5. Johanson KE, Watt TJ, McIntyre NR, Thompson M2013Purification and characterization of enzymes from yeast: an extended undergraduate laboratory sequence for large classesBiochem Mol Biol Educ4125126110.1002/bmb.2070423868379 http://dx.doi.org/10.1002/bmb.20704
6. Manivasakam P, Weber SC, McElver J, Schiestl RH1995Microhomology mediated PCR targeting in Saccharomyces cerevisiaeNucleic Acids Res232799280010.1093/nar/23.14.27997651842307107 http://dx.doi.org/10.1093/nar/23.14.2799
7. Amberg DC, Burke DJ, Strathern JN2006PCR-mediated gene disruption: one-step methodCSH Protoc200610.1101/pdb.prot4169 http://dx.doi.org/10.1101/pdb.prot4169
8. Saraya R, Krikken AM, Kiel JA, Baerends RJ, Veenhuis M, van der Klei IJ2012Novel genetic tools for Hansenula polymorphaFEMS Yeast Res1227127810.1111/j.1567-1364.2011.00772.x http://dx.doi.org/10.1111/j.1567-1364.2011.00772.x
9. Gietz RD, Woods RA2002Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol methodMethods Enzymol350879610.1016/S0076-6879(02)50957-512073338 http://dx.doi.org/10.1016/S0076-6879(02)50957-5
10. Trumbly RJ1992Glucose repression in the yeast Saccharomyces cerevisiaeMol Microbiol6152110.1111/j.1365-2958.1992.tb00832.x1310793 http://dx.doi.org/10.1111/j.1365-2958.1992.tb00832.x
11. Carlson M, Botstein D1982Two differentially regulated mRNAs with different 5′ ends encode secreted with intracellular forms of yeast invertaseCell2814515410.1016/0092-8674(82)90384-17039847 http://dx.doi.org/10.1016/0092-8674(82)90384-1

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2018-10-31
2018-12-18

Abstract:

This article describes a relatively straightforward procedure to knock out the gene that encodes the invertase enzyme in baker’s yeast. The SUC2 gene, which encodes for the invertase enzyme, is knocked out by a single-step PCR knock out method. The knockout is subsequently confirmed at the genetic level by PCR and agarose gel electrophoresis. The knockout is confirmed at the biochemical level by measuring the activity of the invertase enzyme using a colorimetric assay. This tips and tools article describes an easily scalable, inexpensive, yet challenging research project helping undergraduate students at the Bachelor level to conceptualize the effect of the deletion of a gene encoding an enzyme.

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Figures

Image of FIGURE 1

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FIGURE 1

Flowchart of the experiment. Instructor activities are shown in red. Student activities are shown in green. Days are related to student activities. For example: “Practical day 1 – 1 day” means that instructors need to start this activity one day prior to the first practical day in which students participate. WT = Wild-Type; YPD = yeast-extract peptone dextrose.

Source: J. Microbiol. Biol. Educ. October 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1615
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Image of FIGURE 2

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FIGURE 2

Schematic overview of the PCR-mediated single-step gene disruption method. A) Schematic representation of vector pHIPH4 containing the hygromycin B gene (hph). B) The oligonucleotides contain flanking sequences corresponding to the gene. PCR is used to amplify a gene knockout cassette harboring a hygromycin B resistance cassette. Upon transformation in yeast, the target gene will be disrupted by two homologous recombination events at the locus. Clones can be selected for resistance toward hygromycin B. ORF = open reading frame.

Source: J. Microbiol. Biol. Educ. October 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1615
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Image of FIGURE 3

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FIGURE 3

Overview of the data that students are expected to generate. A) The knockout cassette PCR product after agarose electrophoresis. Numbers represent base pairs. B) Hygromycin B–resistant yeast colonies. C) A PCR check for the confirmation of integration at the correct locus. This will show a 687 bp band after agarose electrophoresis. Numbers represent base pairs. Based on the PCR results, clones 1, 2, 4, and 5 are considered knockout whereas clone 3 is considered WT. D) Result of the invertase assay. Red color indicates the presence of reducing sugars. Based on the invertase measurements, clones 1, 2, 4, and 5 are considered knockout whereas clone 3 is considered WT. WT = Wild-Type; bp = base pairs.

Source: J. Microbiol. Biol. Educ. October 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1615
Download as Powerpoint

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