<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>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><b>Aim:</b></p>
<p><b>Aim:</b></p>
<p>To check the reporter signals downstream of the muscone molecular switch.</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>To check the synthesis of the secretion system downstream of the muscone molecular switch.</p>
...
@@ -181,7 +181,6 @@
...
@@ -181,7 +181,6 @@
<divclass="row mt-4">
<divclass="row mt-4">
<divclass="col-lg-12">
<divclass="col-lg-12">
<h3>Muscone molecular switch</h3>
<p>To verify the effectiveness of the muscone molecular switch in <i>Saccharomyces cerevisiae</i>, we used the GFP reporter gene to reflect the signal intensity downstream of the muscone molecular switch. By using the galactose promoter to induce the expression of the muscone molecular switch, we established a glucose-induced control group during the induction process. Additionally, we set up control groups with and without muscone under two different carbon source induction conditions. Details of the induction experiment can be found in the protocol. We captured fluorescence signal images of different groups of <i>Saccharomyces cerevisiae</i> under a confocal microscope and conducted quantitative analysis of relative fluorescence intensity and fluorescence proportion.</p>
<p>To verify the effectiveness of the muscone molecular switch in <i>Saccharomyces cerevisiae</i>, we used the GFP reporter gene to reflect the signal intensity downstream of the muscone molecular switch. By using the galactose promoter to induce the expression of the muscone molecular switch, we established a glucose-induced control group during the induction process. Additionally, we set up control groups with and without muscone under two different carbon source induction conditions. Details of the induction experiment can be found in the protocol. We captured fluorescence signal images of different groups of <i>Saccharomyces cerevisiae</i> under a confocal microscope and conducted quantitative analysis of relative fluorescence intensity and fluorescence proportion.</p>
<p>In the galactose-induced experimental group, the fluorescence intensity and proportion of the GFP reporter gene under muscone induction were significantly higher than those in the control group without muscone. In the glucose control group, there was no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone-induced experimental group and the control group. The experiment preliminarily proves the effectiveness of the introduced muscone molecular switch in <i>Saccharomyces cerevisiae</i>.</p>
<p>In the galactose-induced experimental group, the fluorescence intensity and proportion of the GFP reporter gene under muscone induction were significantly higher than those in the control group without muscone. In the glucose control group, there was no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone-induced experimental group and the control group. The experiment preliminarily proves the effectiveness of the introduced muscone molecular switch in <i>Saccharomyces cerevisiae</i>.</p>
<p>At the same time, we found that compared to the galactose-induced experimental group, the glucose control group showed a higher background noise of mating signals in <i>Saccharomyces cerevisiae</i>. In our subsequent experimental design, we knocked out the original receptor of the <i>Saccharomyces cerevisiae</i> mating signal pathway, which reduced the background signal intensity of the <i>Saccharomyces cerevisiae</i> mating signal pathway and improved the reliability of the system.</p>
<p>At the same time, we found that compared to the galactose-induced experimental group, the glucose control group showed a higher background noise of mating signals in <i>Saccharomyces cerevisiae</i>. In our subsequent experimental design, we knocked out the original receptor of the <i>Saccharomyces cerevisiae</i> mating signal pathway, which reduced the background signal intensity of the <i>Saccharomyces cerevisiae</i> mating signal pathway and improved the reliability of the system.</p>
