RE&Complementation Lab Report

Overexpression plasmids and plated on yc media

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overexpression plasmids and plated on YC media lacking uracil to maintain the plasmids within the transformants. A portion of these transformed cells was set aside and spot plated on YPD media to calculate the number of viable cells and transformation efficiency (O’Connor). Plasmid 14 had the greatest percent transformed and transformation efficiency of all of the yeast overexpression plasmids, probably because it contains the same gene that was originally deleted in the met3 auxotroph. Plasmid 4 had the next greatest transformation efficiency, while plasmid 10 had the least efficient transformation (Table 2). Yeast cells transformed on YC-Ura media underwent replica plating and complementation analysis on various media and significant growth occurred for pYES2.1+ Met3 and pBG1805+ MET3 without methionine (Fig. 2). The yeast cells transformed on YC-Ura media contain a GAL1 promoter, which represses fusion protein expression in the presence of glucose and induces fusion protein expression in the presence of galactose. In the presence of galactose, pYES2.1+ Met3, which contained an orthologous MET3 gene, and pBG1805+ MET3 were able to complement the met3 auxotrophy and allow yeast growth in the absence of methionine. No growth occurred in any transformed cells in the YC + glu – met – ura media because glucose represses the GAL1 promoter. However, growth would be
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expected in YC + glu + met – ura, because the media provides methionine that allows the transformed S. cerevisiae cell to grow even with a repressed GAL1 promoter. Baseline transcription occurred in met3 auxotrophs transformed with the pYES2.1- lacZ overexpression plasmid, but this was most likely due to a leaky promoter. Yeast cells transformed with pYES2.1- lacZ were not expected to grow because successful complementation depends on the expression of the fusion protein and the ability of the fusion protein to restore enzymatic activity that is missing in the mutant strain. The pYES2.1- lacZ overexpression plasmid does not contain a MET3 homologue or ortholog, and even if its fusion protein were expressed it would not restore the cells ability to grow; glycolysis could not occur and cells could not grow. Growth with pYES2.1+ Met3 and pBG1805+ MET3 occurs because Met3, also known as sua1, is an ortholog of MET3 and was probably conserved during evolution considering its vital role in cell growth. A pairwise alignment of Met3p and the Saccharomyces pombe ortholog, sulfate adenylyltransferase, has an
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Christopher Reinemann
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