Single-Step Gene Knockout of the SUC2 Gene in Saccharomyces cerevisiae : A Laboratory Exercise for Undergraduate Students †
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Authors:
Jurre Hageman1,*,
Arjen M. Krikken2
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Received 13 April 2018 Accepted 11 July 2018 Published 31 October 2018
- ©2018 Author(s). Published by the American Society for Microbiology.
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[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.
- *Corresponding author. Mailing address: Expertise Centre ALIFE, Institute for Life Science & Technology, Hanze University of Applied Sciences, Groningen, Groningen, The Netherlands. Phone: +31 505954569. E-mail: [email protected].
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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
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
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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Supplemental Material
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Appendix 1: PDF file of the protocol for students, Appendix 2: PDF file with instructor recourses
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2018-10-31
2019-10-21
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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Author and Article Information
-
Received 13 April 2018 Accepted 11 July 2018 Published 31 October 2018
- ©2018 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.
- *Corresponding author. Mailing address: Expertise Centre ALIFE, Institute for Life Science & Technology, Hanze University of Applied Sciences, Groningen, Groningen, The Netherlands. Phone: +31 505954569. E-mail: [email protected].
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Single-Step Gene Knockout of the SUC2 Gene in Saccharomyces cerevisiae : A Laboratory Exercise for Undergraduate Students
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Citation:
Hageman J, Krikken A. 2018. Single-step gene knockout of the suc2 gene in saccharomyces cerevisiae : a laboratory exercise for undergraduate students
†
. J. Microbiol. Biol. Educ. 19(3): doi:10.1128/jmbe.v19i3.1615
/content/jmbe/10.1128/jmbe.v19i3.1615.f1-jmbe-19-92
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.
jmbe/19/3/jmbe-19-92f1_thmb.gif
jmbe/19/3/jmbe-19-92f1.gif
Click to view
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
/content/jmbe/10.1128/jmbe.v19i3.1615.f2-jmbe-19-92
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 SUC2 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 SUC2 locus. Clones can be selected for resistance toward hygromycin B. ORF = open reading frame.
jmbe/19/3/jmbe-19-92f2_thmb.gif
jmbe/19/3/jmbe-19-92f2.gif
Click to view
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 SUC2 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 SUC2 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
/content/jmbe/10.1128/jmbe.v19i3.1615.f3-jmbe-19-92
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 SUC2 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 SUC2 knockout whereas clone 3 is considered WT. WT = Wild-Type; bp = base pairs.
jmbe/19/3/jmbe-19-92f3_thmb.gif
jmbe/19/3/jmbe-19-92f3.gif
Click to view
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 SUC2 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 SUC2 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
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