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+
+<div class="row mt-4">
+  <div class="col-lg-12">
+    <h2 id="Results">
+      <h2>Results</h2>
+      <hr>
+    <p>Through our efforts, we have obtained some experimental results to support our system design. We divided the therapy system into an upstream muscone molecular signal switch and a downstream lactate secretion system, and we have tested their operational effectiveness through a series of wet lab experiments. Finally, we integrated them to test the effectiveness of the complete therapy system. You can see our experimental results in the following content:</p>  
+</div>
+</div>
+
+<div class="row mt-4">
+  <div class="col-lg-12">
+    <h3>Muscone molecular switch</h3>
+    <p>In our system, the muscone molecule serves as the signal controlling the secretion system. Introducing the muscone molecular signal switch into the signaling pathway of Saccharomyces cerevisiae is one of our core tasks. You can view our experimental design through the design.</p>
+  </div>
+</div>
+
+<div class="row mt-4">
+  <div class="col-lg-12">
+    <h3>Muscone molecular switch</h3>
+    <p>To verify the effectiveness of the muscone molecular switch in Saccharomyces cerevisiae, 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 Saccharomyces cerevisiae 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 Saccharomyces cerevisiae.</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 Saccharomyces cerevisiae. In our subsequent experimental design, we knocked out the original receptor of the Saccharomyces cerevisiae mating signal pathway, which reduced the background signal intensity of the Saccharomyces cerevisiae mating signal pathway and improved the reliability of the system.</p>
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig8.png" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 8 Muscone molecular switch fluorescence signal test, A. Galactose-induced, add muscone organic solution. B. Galactose-induced, without muscone. C. Glucose control group, add muscone organic solution. D. Glucose control group, without muscone.</p>
+    </div>
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig9.jpg" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 9 Quantitative analysis of muscone molecular switch fluorescence signal.</p>
+    </div>
+  </div>
+</div>
+
+<div class="row mt-4">
+  <div class="col-lg-12">
+    <h3>Lactate secretion</h3>
+    <p>In our system, the lactic acid molecule is the small molecule that ultimately treats IBD diseases and alleviates the abnormal activation of autoimmune cells. We have altered the anaerobic metabolic pathway of Saccharomyces cerevisiae by inducing the expression of lactate dehydrogenase from E. coli with galactose, and tested the effectiveness of the secretion system. You can view our experimental design through the design.</p>
+    <p>We used the galactose promoter to control the expression of lactate dehydrogenase within Saccharomyces cerevisiae. We established a glucose-induced control group during the induction process. And we also conducted an induction experiment with untransformed wild-type yeast as a control to exclude the background signal noise of Saccharomyces cerevisiae. Details of the induction experiment can be found in the protocol. After induction, the bacterial solution was centrifuged, and the supernatant was collected. The WST colorimetric method was used to measure the lactic acid content in the supernatant of different groups. The absorbance of the colorimetric reaction was recorded at 455 nm, which reflects the concentration of D-lactic acid in the supernatant.</p>
+    <p>The experimental results preliminarily demonstrate that in the transformed groups, galactose induction led to the expression of lactate dehydrogenase, which successfully altered the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize D-lactic acid. Meanwhile, after the synthesis of D-lactic acid, it can be secreted into the surrounding environment of the Saccharomyces cerevisiae.</p>
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig10.png" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 10 Galactose-induced D-lactic acid secretion system</p>
+    </div>
+    <p>Taking into account that different carbon sources for induction and cultivation systems may affect the growth of Saccharomyces cerevisiae and the working rate of lactate dehydrogenase. We further established different combinations of induction and cultivation with glucose and galactose groups based on previous experiments, and set up a time gradient to sample the supernatant of Saccharomyces cerevisiae cultures for the measurement of D-lactic acid concentration.</p>
+    <p>The experimental results show that, compared to the galactose group, the glucose group has a non-specific promotional effect on the secretion of D-lactic acid by Saccharomyces cerevisiae. In the short term of induction and cultivation, glucose will synthesize more D-lactic acid by promoting yeast growth more strongly and accelerating the reaction rate of lactate dehydrogenase. However, in longer-term induction experiments, the galactose group will ultimately synthesize more D-lactic acid due to the specific induction of lactate dehydrogenase expression. This experimental result confirms the impact of different carbon source cultivation systems on the secretion system. To eliminate unnecessary influences as much as possible, we adopted a muscone molecular switch expressed by a constitutive promoter in subsequent experiments, removing the galactose-induced experimental step.</p>
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig13.png" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 11 Changes in lactic acid secretion with different carbon source combinations in short-term culture.</p>
+    </div>
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig11.jpg" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 12 Changes in lactic acid secretion with different carbon source combinations in long-term culture.</p>
+    </div>
+  </div>
+</div>
+
+<div class="row mt-4">
+  <div class="col-lg-12">
+    <h3>Origin receptor knock-out</h3>
+    <p>Based on the previous experimental results of the muscone molecular switch signal, we designed a knockout system targeting the original receptor STE2 of the mating pathway in Saccharomyces cerevisiae, including the gRNA and Cas9 protein targeting STE2. We performed sequencing on the successfully transformed strains and confirmed that base deletions and frameshift mutations occurred in the original receptor STE2 sequence. We retransformed the strains with successful STE2 knockout with MOR215&Ga-pESC and pFUS1 promoter-GFP-pYES and repeated the previous induction experimental protocol. The results of the muscone analysis switch signal intensity in the knockout Saccharomyces cerevisiae strain were obtained under a confocal microscope.</p>
+    <p>The experimental results show that under the muscone induction condition in the galactose group, the fluorescence intensity and proportion of the GFP reporter gene remained significantly higher than that of the control group without muscone. In the glucose group, there was still no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone induction group and the control group without muscone. Compared to the strain with the original receptor STE2 not knocked out, the background signal noise in the glucose group significantly decreased after the original receptor STE2 was knocked out. The experiment confirmed that knocking out the original receptor STE2 of the Saccharomyces cerevisiae mating pathway reduced the noise of the background mating signal, thus improving the reliability of the muscone molecular switch signal.</p>
+  
+    <div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
+      <img src="https://static.igem.wiki/teams/5187/wiki-therapysystem-fig/fig12.jpg" alt="ibd_figure" class="shadowed-image" style="width: 45%; max-width: 400px;">
+      <p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 13 Quantitative analysis of muscone molecular switch fluorescence signal of knocking out STE2 strain</p>
+    </div>
+   
+  </div>
+</div>
+
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