A Metabolic Bait-and-Switch: Deleting the ADE2 Gene by Replacing it with HIS3

Lab report written based on experimentation performed in my Molecular Genetics Laboratory, gene editing Saccharomyces cerevisiae using Arabidopsis thaliana.

Introduction
Saccharomyces cerevisiae, colloquially known as bakers yeast, is a workhorse of molecular genetics research. As a single-celled eukaryotic organism the ease at which it can be grown and studied is similar to bacterial organisms, with the added benefit that “nearly all biological functions found in eukaryotes are also present and well conserved in S. cerevisiae” (Parapouli et al., 2020). Targeted mutagenesis refers to the deletion of a specific gene from the chromosome, producing a knock out organism whose phenotype helps in determining the function of the deleted gene. This type of mutagenesis is most easily performed in single-celled organisms, because cells possessing the induced mutation can easily be isolated and observed. Especially in S. cerevisiae, this is often performed by utilizing homologous recombination to replace the endogenous target gene (Hegemann et al., 2014). Researchers are working to systematically disrupt every ORF in the S. cerevisiae genome, performing targeted mutagenesis to identify the function of all yeast genes. In this experiment, the ADE2 gene was targeted because of detailed knowledge of the upstream and downstream sequences, which allows for the selection/creation of a plasmid that has the homology needed to be inserted into the gene at the correct location.
In order to facilitate the deletion of the ADE2 gene in yeast strain BY4742, the pRS403 plasmid was prepared and a fragment containing regions homologous to the upstream and downstream regions of the ADE2 gene was amplified using PCR. This fragment also possesses the HIS3 gene which functions as a selectable marker present in the transformants produced. Transformants were then characterized by their phenotype, because a red phenotype indicates disruption in the adenine synthesis pathway. We postulated that homologous recombination of an inserted HIS3 and the existing ADE2 gene will result in the deletion of the ADE2 gene, identifiable by the red phenotype of transformants on plates with low adenine supplementation.
Results
pRS403 was successfully recovered from E. coli cells as evidenced by nanodrop spectrophotometry seen in Figure 1. The measured concentration of 34.4 ng/ul was used to prepare the PCR reaction used to amplify the fragment of interest, who’s presence was tested for in Figure 2. PCR was performed on three primer preps (one for each group member) of which two (myself and one other) were successful, indicated by the bands at approximately 1232 bp, which is the expected size of the HIS3 gene and the sequences needed for homologous recombination.
Strain BY4742 was then transformed using the PCR product, and transformants were selected for by plating onto histidine dropout media containing a small amount of adenine. Of these transformants, some grew with a red phenotype and others with a white phenotype. Table 1 summarizes the number of red and white colonies on a representative plate, used to determine the gene deletion frequency of 2%. PCR was then used to confirm deletion of the ADE2 gene in a representative red and white colony through the use of 2 distinct primer pairs. The subsequent gel electrophoresis of these PCR products can be seen in Figure 3.
Discussion
Preparation of the pRS403 plasmid was successful, in that a sample of high concentration with good DNA purity was obtained (Figure 1). The DNA purity is measured by the 260/280 ratio, with a high ratio (1.85) indicating there is more DNA than protein, which is the desired result. PCR of the plasmid should have resulted in a band at ~1232 bp, representing the total length of the fragment of interest: the HIS3 gene and two 20-nucleotide long sequences which enable homologous recombination. This expected product was seen in lanes 2 and 4 (Figure 2), indicating successful PCR amplification. Lane 3, however, did not possess any bands. The plasmid loaded into lane 3 was successfully prepped, so this lack of a band is likely due to an issue in PCR setup (such as a missing reagent). The success of transformation of the strain BY4742 with the pRS403 plasmid product was tested by plating onto histadine dropout plates which select for those cells containing the HIS3 gene. These plates only included enough adenine to facilitate the growth of the cells while still keeping the ADE2 pathway activated, allowing for the expression of HIS3. The histidine dropout plates grew red and white colonies, a representative plate is summarized in Table 1. A 2% gene deletion frequency was expected and achieved, indicating the experiment was successful. PCR was then performed using 4F/5R and 6F/7R primer pairs (Figure 3) to confirm the deletion of the ADE2 gene in the red colonies. The 4F/5R primer pair should amplify fragments in colonies with a functional adenine biosynthesis pathway (the white colonies), while the 6F/7R primers should amplify fragments between the ADE2 flanking sequences and the HIS3 coding regions in colonies where gene deletion of ADE2 using HIS3 was successful (the red colonies). These expected results were reflected in gel electrophoresis in Figure 3, where the 6F/7R HIS3 band is in lane 2, in the red colony, at the approximate length of the amplified region (~448 bp) and the 4F/5R ADE2 band is in lane 4, in the white colony, at the approximate length of the amplified region (~319 bp).
Our hypothesis was correct. The HIS3 gene, via targeted mutagenesis of the ADE2 gene, successfully resulted in the deletion of the ADE2 gene and subsequent loss of ADE2 function, resulting in the colonies exhibiting a red phenotype. Performing PCR and gel electrophoresis on multiple red and white colonies (as opposed to one representative colony of each) would allow for a more strongly supported conclusion that the deletion and homologous recombination were successful. A similar experiment could be performed targeting a gene other than ADE2, which would require new plasmids and primers but would follow the same theoretical procedure of this experiment.
Works Cited
Hegemann, J. H., Heick, S. B., Pöhlmann, J., Langen, M. M., & Fleig, U. (2014). Targeted Gene Deletion in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In Methods in molecular biology (pp. 45–73). https://doi.org/10.1007/978-1-4939-0799-1_5 
Parapouli, M., Vasileiadis, A., Afendra, A. S., & Hatziloukas, E. (2020). Saccharomyces cerevisiae and its industrial applications. AIMS Microbiology, 6(1), 1–32. https://doi.org/10.3934/microbiol.2020001