Single-Step Gene Knockout of the SUC2 Gene in Saccharomyces cerevisiae : A Laboratory Exercise for Undergraduate Students†

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.


Yeast as a model organism
Yeast are attractive organisms to study. Yeast are single-celled eukaryotic organisms that are easy to grow in the laboratory and yeast cells can be maintained as a haploid organism which strongly simplifies genetics. Saccharomyces cerevisiae (see figure 1) has been used in winemaking, brewing and baking since ancient times. More recently, Saccharomyces cerevisiae has been studied extensively in the laboratory and is now considered a model organism to study the behavior of eukaryotic cells such as mitosis and general metabolism. Various genetic techniques are readily applicable in Saccharomyces cerevisiae. Especially the generation of knock-out strains is extremely powerful in Saccharomyces cerevisiae. A gene knock-out can be achieved within a week which greatly enhances the speed of understanding gene function. Because of a high degree of similarity between yeast genes and their human orthologues, yeast has become a popular model system for the study of human genetic disease 1 .

Biology and reproduction cycle of Saccharomyces cerevisiae
Yeast are single-celled eukaryotes that belong to the kingdom Fungi. Because yeast are eukaryotes, they harbor a similar basic cellular organization as can be seen in more complex eukaryotic cells such as human cells. However, the organization of the genome is much simpler. Intronic sequences, for instance, are very rare in the genome of Saccharomyces cerevisiae. Saccharomyces cerevisiae is a budding yeast and belongs to the phylum Ascomycota 1 .
When cultured as a haploid organism, Saccharomyces cerevisiae grows asexually by budding. Yeast cells divide by mitosis. A small bud grows on the mother cell. The mother cell duplicates its chromosomes which are segregated into the daughter cell. Meanwhile the daughter cell grows, a nucleus will form and eventually the daughter cell buds off from the mother cell. All classic mitotic stages (G1, S, G2 and M) as well as cytokinesis can be distinguished 2 .
Beside asexual growth, Saccharomyces cerevisiae can grow sexually. When in close proximity and when growth conditions are unfavorable, haploid yeast cells can mate. The cells and nuclei will fuse and the resulting cell is now diploid. These diploid cells can reproduce asexually by budding, similar to haploid cells as described above. However, when growth conditions start to become unfavorable, the cells undergo sporulation. During this stage, the cells will undergo meiosis. Homologous chromosomes will align and during meiosis I the homologous chromosomes will separate. These cells contain a single chromosome complement (1n) but the chromosomes still consist of sister chromatids. During meiosis II, sister chromatids will separate. Eventually, four spores will be generated which can survive long periods of time. When conditions become more favorable, the spores are able to germinate and reproduce asexually. The life cycle of Saccharomyces cerevisiae is summarized in figure 2 3 .

Mating types
Saccharomyces cerevisiae is heterothallic which means that each yeast cell is of a certain mating type. Two different mating types can be distinguished: the a mating type and the alpha mating type. Only cells of opposite mating types can mate (conjugate) and this phenomenon can be seen as a simple form of sexual differentiation. In the laboratory, mating is often not desired to take place as it increases the complexity of genetics. During many experiments, it is desired to culture the yeast cells as haploid cells that reproduce asexually. Keeping one type of cell (a or alpha) is normally not feasible as yeast cells can undergo mating type switching that changes the mating type from a to alpha and vice versa. However, mutant laboratory strains have been generated that are deficient in mating type switching. This ensures the culture of solely haploid cells via asexual reproduction 3 .

Gene manipulation in Saccharomyces cerevisiae: generating a knock-out strain
Saccharomyces cerevisiae is very well studied and amenable to genetic manipulation. A key feature of Saccharomyces cerevisiae is its efficient homologous recombination. Homologous recombination is very precise in double strand break repair compared to non-homologous end joining. The efficient homologous recombination in Saccharomyces cerevisiae is widely exploited to generate specific (targeted) knock-out strains 4 . This technological breakthrough has increased our understanding on the function of genes tremendously. For the generation of a gene knock-out, one can simply clone two yeast sequences that are in close proximity to each other in the genome around a marker gene (such as an antibiotic marker) and integration of the marker gene will almost always take place at the genomic site homologous to the cloned sequences. The marker can be used to select the transformants 4 . A technical improvement to the above method is to use a one-step PCR method in which the "yeast insertion sequences" are designed as oligonucleotide sequences 5 . This eliminates the laborious cloning step. The oligonucleotide sequences can be used as primers to amplify an antibiotic resistance (Hygromycin) gene cassette (containing yeast promoter and terminator sequences) from a plasmid (pHIPH4). After transformation, homologous recombination as double cross-over will replace the open reading frame of the gene of interest. Selection of antibiotic resistant colonies will select for transformants. PCR can be used to verify the integration. Thereafter the gene function can be studied with functional (biochemical) assays. See figure 3 for an outline of the one-step PCR knock-out technology. Because yeast can be grown as a haploid organism, there is only a single copy (allele) that simplifies the generation of a knock-out strain.

Invertase
Invertase is an enzyme encoded by the SUC2 gene that catalyzes the hydrolysis of table sugar (sucrose) to glucose and fructose:

Sucrose -> Glucose + Fructose
Yeast use invertase to digest sucrose into monosaccharides that can be further degraded by glycolysis and the Krebbs cycle to conserve energy in the form of ATP. The expression of invertase is strongly repressed by high concentrations of glucose 6,7 .

Goal of the experiment
The goal of this experiment is to generate a gene-knockout in Saccharomyces cerevisiae of the invertase gene. The integration of the antibiotic gene at the correct locus will be experimentally verified by measuring the invertase activity using a biochemical assay. 5. Saccharomyces cerevisiae is a powerful model organism as the generation of a gene knock-out is relatively easy. Describe the one-step knock-out strategy in your own words.

Reagents and media
YPD will be prepared by the institute and supplied by your supervisor. Reagents: -Bacto agar (if preparing agar plates) -Bacto peptone -Glucose (40%, w/v). sterilize by filtration or autoclaving.
-Yeast extract (Fisher Scientific) Procedure:  To an autoclavable flask, add: Reagent Amount to add (per 1 L final volume) Liquid Agar plates Bacto agar --24 g Bacto peptone 20 g 20 g Yeast extract 10 g 10 g Water 950 mL 950 mL  Autoclave the mixture.  Add 50 mL of sterile 40% (w/v) glucose. Mix.  Allow to cool before use.

PCR (Day 1)
Set up a series of PCR reactions. Include a negative control simulate all PCR reactions and record the expected product lengths in your lab journal. Note: the information below lacks many details. Prepare a detailed pipetting scheme before you start! Calculate how much of each ingredient you need. Also, set up a negative control PCR using water instead of template DNA. For PCR conditions see the manufactures protocol (TEMPase Hot Start DNA Polymerase PCR, VWR chemicals). Use 25 ng of plasmid (pHIPH4) DNA for a PCR reaction. Concentration of the stock is 25 ng/µl. Prepare a stock of the dNTP mix. Stock solution of each separate dNTP is 100 mM. Stock of the combined dNTP solution should be 10 mM for each dNTP (40 mM combined). PCR conditions: Denaturation for 1 minute at 95˚C followed with 30 cycles of 95˚C for 30 seconds, 58˚C for 40 seconds and 72˚C for 2 minutes. Add a final extension step of 5 minutes at 72˚C. Primers will be provided as 10 µM in sterile water. Primer sequences: KO-SUC2-f: ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCCCACACACCATAGCTTCAA KO-SUC2-r: CTATTTTACTTCCCTTACTTGGAACTTGTCAATGTAGAACCGTTTTCGACACTGGATGGC

Agarose gel electrophoresis (Day 2)
Check the product sizes by agarose gel electrophoresis in 1x TBE. Do not forget to run an appropriate marker. Choose the agarose concentration based on the expected product sizes. Use Midori green at the appropriate concentration as a DNA staining dye in the gel. Visualize your gel in a chemidoc and save the pictures.

Transformation (Day 1: preparing cells, Day 2: transformation)
Solutions: All these solutions will be prepared by the institute and supplied by your supervisor except for the Hygromycin plates. You need to prepare these yourself!  Sterile water: autoclave MQ water  Herring sperm (or Salmon sperm) DNA (2 mg/ml). Denature prior to use by keeping it at 100 °C for 10 min and cooling it immediately by placing on icewater.  40% PEG 3350 in LiAc/TE buffer: 50% PEG in water 8 ml 100mM Tris-Hcl pH 7.5, 10mM EDTA 1 ml 1 M Lithium acetate 1 ml  1 M Sorbitol in water  YPD (see above)  20 mg/ml Hygromycin: dissolve Hygromycin at 20 mg/ml and filter sterilize  YPD plates with hygromycin: Add 1.5% agar to 100 ml YPD (prepare this YPD yourself), autoclave. When ~ 50 ˚C, add hygromycin to a final concentration of 200 µg/ml. Pour approximately 5 agar plates from the YPD agar.
Procedure:  Inoculate yeast cells in 20 ml YPD liquid media and incubate overnight at 30 °C at 220 rpm rotation. This step is already completed by your supervisor. DAY1  Measure the OD 600 of the overnight culture and dilute it to OD600 of 0.25 in 50 ml YPD  Grow cells until the OD600 is 0.8 -1.0. This will take approximately 3 hours.  Pick 6 colonies (transformants) and grow in 2 ml YPD overnight to saturation at 30˚C at 220 rpm. Grow a wild-type strain in YPD (negative control).  Pellet 1.5 ml of cells for 2 min. at 10,000 rpm in an eppendorf centrifuge.  Transfer the rest of the culture medium to a sterile 50 ml tube containing 10 ml of fresh YPD culture medium and incubate overnight at 30˚C at 220 rpm. This will be used for the invertase assay (day 5).  Resuspend cells in 500 µl T 100E50 (pH 8) + 5 µl β-mercaptoethanol.  Primer sequences are: forward check primer: 5'-GTCGCCAACATCTTCTTCTG-3', reverse check primer, 5'-GCTAAAGCCCTTTAGAATGG-3'.  For the invertase knock-out, use the same PCR protocol as above.  Mix 10 µl of the PCR product with 2 µl of loading dye and run on an 1.5% (w/v) agarose gel.
If short in time, this step can be performed at day 5.

Measure activity (Day 5 )
You can investigate if your gene-knock out was functionally successful by setting up a biochemical experiment to measure the activity of the knock-out gene product. To confirm the invertase knockout, we will use an invertase assay. Invertase activity can be measured indirectly using 3,5dinitrosalicylic acid (DNS) reagent. DNS is an aromatic compound that reacts with reducing sugars to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. Thus, the absorbance at 540 nm is a measure of the amount of glucose and fructose formed by the invertase activity. The expression of the invertase gene is repressed by high concentrations of glucose. Therefore, cells should by incubated at low glucose concentrations (YPD with 0,05% glucose instead of 2% glucose called YPlowD).

Method: DAY5:
 Wash the cells with 10 ml sterile water (centrifuge 4000 rpm for 5 min., resuspend in 10 ml of sterile water and centrifuge 4000 rpm for 5 min. again).  Resuspend in 10 ml of YP-low-D (0,05% glucose).  Culture for 3 hours at 30˚C, 220 rpm.  Centrifuge 4000 rpm for 5 min.  wash once with 10 ml sterile water.  Dissolve pellet in 1 ml sterile water and transfer to 1,5 ml vial.  Centrifuge 4000 rpm for 5 min.  Add 0,5 ml of sterile water and freeze at -80˚C.  Freeze/thaw another round. For the assay: Use water instead of sucrose as negative control. Use 1 ml of 1% glucose instead of sucrose as positive control.
 Add 1 ml sucrose 0.25 M to a tube (water for negative control, 2 ml of 1% glucose for positive control.  Add 1 ml ddH2O.  Add 0,9 ml 0,05 M sodium acetate (pH 4,7).  Add 0.1 ml cell extract.  Incubate 60 min. at 40˚C.  Add 1 ml DNS reagent.  Incubate 10 min. at 100˚C.  Cool fast until room temperature.  Measure the OD at 540 nm and make a picture of the tubes.