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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 R 1991 Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast Methods Enzymol 194 281 301 10.1016/0076-6879(91)94022-5 2005793 http://dx.doi.org/10.1016/0076-6879(91)94022-5
2. Wendland J 2003 PCR-based methods facilitate targeted gene manipulations and cloning procedures Curr Genet 44 115 123 10.1007/s00294-003-0436-x 12928752 http://dx.doi.org/10.1007/s00294-003-0436-x
3. Hinnebusch AG, Johnston M 2011 YeastBook: an encyclopedia of the reference eukaryotic cell Genetics 189 683 684 10.1534/genetics.111.135129 22084419 3213385 http://dx.doi.org/10.1534/genetics.111.135129
4. Myrback K 1957 Studies on yeast invertase; soluble and insoluble invertase (saccharase) of baker’s yeast Arch Biochem Biophys 69 138 148 10.1016/0003-9861(57)90481-2 13445188 http://dx.doi.org/10.1016/0003-9861(57)90481-2
5. Johanson KE, Watt TJ, McIntyre NR, Thompson M 2013 Purification and characterization of enzymes from yeast: an extended undergraduate laboratory sequence for large classes Biochem Mol Biol Educ 41 251 261 10.1002/bmb.20704 23868379 http://dx.doi.org/10.1002/bmb.20704
6. Manivasakam P, Weber SC, McElver J, Schiestl RH 1995 Microhomology mediated PCR targeting in Saccharomyces cerevisiae Nucleic Acids Res 23 2799 2800 10.1093/nar/23.14.2799 7651842 307107 http://dx.doi.org/10.1093/nar/23.14.2799
7. Amberg DC, Burke DJ, Strathern JN 2006 PCR-mediated gene disruption: one-step method CSH Protoc 2006 10.1101/pdb.prot4169 http://dx.doi.org/10.1101/pdb.prot4169
8. Saraya R, Krikken AM, Kiel JA, Baerends RJ, Veenhuis M, van der Klei IJ 2012 Novel genetic tools for Hansenula polymorpha FEMS Yeast Res 12 271 278 10.1111/j.1567-1364.2011.00772.x http://dx.doi.org/10.1111/j.1567-1364.2011.00772.x
9. Gietz RD, Woods RA 2002 Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method Methods Enzymol 350 87 96 10.1016/S0076-6879(02)50957-5 12073338 http://dx.doi.org/10.1016/S0076-6879(02)50957-5
10. Trumbly RJ 1992 Glucose repression in the yeast Saccharomyces cerevisiae Mol Microbiol 6 15 21 10.1111/j.1365-2958.1992.tb00832.x 1310793 http://dx.doi.org/10.1111/j.1365-2958.1992.tb00832.x
11. Carlson M, Botstein D 1982 Two differentially regulated mRNAs with different 5′ ends encode secreted with intracellular forms of yeast invertase Cell 28 145 154 10.1016/0092-8674(82)90384-1 7039847 http://dx.doi.org/10.1016/0092-8674(82)90384-1

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2018-10-31
2019-02-19

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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