<p>We selected muscone gas molecules as the upstream control signal for our therapy system. We used the muscone receptor sequence from mouse olfactory epithelial cells, as employed by the Ye Haifeng team <sup>[1]</sup>; for details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank"style="color: #FF5151 ;">description</a>, and introduced it into the plasmid system expressed in <i>Saccharomyces cerevisiae</i>. We chose the mating pathway in <i>Saccharomyces cerevisiae</i> as the transmission pathway for the muscone signal within <i>Saccharomyces cerevisiae</i>. Based on the Benjamin M Scott team's optimization<sup>[2]</sup>, we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway, allowing the muscone receptor to be integrated into the <i>Saccharomyces cerevisiae</i> mating pathway.</p>
<p>We used the galactose promoter to induce the expression of the muscone signal receptor and the optimized Gα protein, and screened the successfully transformed <i>Saccharomyces cerevisiae</i> with a His nutritional deficiency. By controlling the induction conditions of galactose and muscone, we tested the effectiveness of the muscone gas molecule switch. For details, please refer to the protocol.</p>
<p>Aim:</p>
<p><b>Aim:</b></p>
<p>To validate the effectiveness of the muscone gas molecule switch in <i>Saccharomyces cerevisiae</i>.</p>
<p>We chose the mating pathway in <i>Saccharomyces cerevisiae</i> as the conduit for muscone signaling in yeast. Using the mating pathway’s pFUS1 promoter, we expressed the downstream lactate dehydrogenase to alter the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i>, secreting lactic acid for the treatment of IBD<sup>[3]</sup>. Initially, we designed a plasmid with the pFUS1 promoter expressing the GFP reporter gene and screened the successfully transformed yeast using Ura nutritional deficiency. We then tested the effectiveness of the muscone molecular switch using confocal microscopy; for details, please refer to the protocol. Subsequently, we designed the pFUS1 promoter to express lactate dehydrogenase from E. coli. By co-transforming it with Muscone Receptor & Gα (pESC) into <i>Saccharomyces cerevisiae</i>, we achieved the construction of the complete pathway.</p>
<p>Aim:</p>
<p><b>Aim:</b></p>
<p>To check the reporter signals downstream of the muscone molecular switch.</p>
<p>To check the synthesis of the secretion system downstream of the muscone molecular switch.</p>
<p>Lactic acid is the small molecule we selected to target the abnormally activated autoimmune cells in IBD diseases. For details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank">description</a>. We chose lactate dehydrogenase from E. coli to alter the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> to synthesize and secrete D-lactic acid. We used the galactose promoter to induce the expression of lactate dehydrogenase and screened the successfully transformed yeast with a Ura nutritional deficiency. By controlling the induction with galactose and glucose and establishing gradients of induction time and post-induction culture time, we tested the synthesis and secretion of lactic acid and searched for the optimal induction conditions for lactic acid secretion. For details, please refer to the protocol.</p>
<p>Aim:</p>
<p><b>Aim:</b></p>
<p>To test the effectiveness of the lactate secretion system in <i>Saccharomyces cerevisiae</i>.</p>
<p>To explore the optimal induction conditions for the lactate secretion system.</p>
