<p>In therapy system, we have incorporated muscone-sensing receptors, derived from mouse olfactory epithelial cells, into Saccharomyces cerevisiae. These receptors, which are G protein-coupled receptors (GPCR) in eukaryotic cells, have been integrated into the yeast's signaling pathways. By altering the mating pathway of Saccharomyces cerevisiae, we enabled the muscone receptors to function within this microbial chassis. Additionally, we introduced lactate dehydrogenase downstream of the modified mating pathway, thereby redirecting the yeast's anaerobic metabolism to produce lactate, which is intended for the treatment of Inflammatory Bowel Disease (IBD).</p>
<p>In therapy system, we have incorporated muscone-sensing receptors, derived from mouse olfactory epithelial cells, into <i>Saccharomyces cerevisiae</i>. These receptors, which are G protein-coupled receptors (GPCR) in eukaryotic cells, have been integrated into the yeast's signaling pathways. By altering the mating pathway of <i>Saccharomyces cerevisiae</i>, we enabled the muscone receptors to function within this microbial chassis. Additionally, we introduced lactate dehydrogenase downstream of the modified mating pathway, thereby redirecting the yeast's anaerobic metabolism to produce lactate, which is intended for the treatment of Inflammatory Bowel Disease (IBD).</p>
<p>To better construct the system of muscone-induced lactate secretion, we split it into two parts: one is lactate secretion, and the other is the muscone switch. We merged the two parts together after confirming that both parts can work normally. This project is divided into three main cycles.</p>
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<h2id="Colonization System">
<h2>Colonization System</h2>
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<p>In order to better construct the colonization system that enables Saccharomyces cerevisiae to colonize IBD lesions, we have divided it into two sub-parts: one is the IBD signaling molecule sensor, and the other is the adhesion protein. Only after both parts are confirmed to be functioning properly, we will integrate these two sub-parts together. This part is divided into two main loops. </p>
<p>In order to better construct the colonization system that enables <i>Saccharomyces cerevisiae</i> to colonize IBD lesions, we have divided it into two sub-parts: one is the IBD signaling molecule sensor, and the other is the adhesion protein. Only after both parts are confirmed to be functioning properly, we will integrate these two sub-parts together. This part is divided into two main loops. </p>
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<h3>Big circle1: Tetrathionate sensor</h3>
<p>To enable Saccharomyces cerevisiae to “sense” the presence of IBD, we need a sensor for IBD. Through literature research, we have found that the concentration of tetrathionate in the intestine can characterize the degree of IBD. Additionally, there is current research that has constructed a tetrathionate sensor TtrSR in E. coli. Therefore, we have decided to introduce the tetrathionate sensor TtrSR into yeast cells, using it as a sensor for IBD.</p>
<p>To enable <i>Saccharomyces cerevisiae</i> to “sense” the presence of IBD, we need a sensor for IBD. Through literature research, we have found that the concentration of tetrathionate in the intestine can characterize the degree of IBD. Additionally, there is current research that has constructed a tetrathionate sensor TtrSR in E. coli. Therefore, we have decided to introduce the tetrathionate sensor TtrSR into yeast cells, using it as a sensor for IBD.</p>
<h4>Cycle1</h4>
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<h5>Design</h5>
<p>We have decided to introduce the tetrathionate sensor TtrSR into Saccharomyces cerevisiae cells, utilizing it as a detector for IBD. To test its effectiveness within the cell, we will express EGFP downstream and measure its expression level through fluorescence intensity.</p>
<p>We have decided to introduce the tetrathionate sensor TtrSR into <i>Saccharomyces cerevisiae</i> cells, utilizing it as a detector for IBD. To test its effectiveness within the cell, we will express EGFP downstream and measure its expression level through fluorescence intensity.</p>
<h5>Build</h5>
<p>After confirming the sequence, we designed plasmids pECS and pYES2 that can be used to construct the tetrathionate sensor TtrSR system in brewing yeast. These plasmids carry the URA3 and HIS3 genes, respectively, as selection markers. figures below show the details.</p>
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<h5>Learn</h5>
<p>After optimizing the protocol, we successfully observed green fluorescence. The experimental results showed that the expression of the downstream protein EGFP increased after tetrathionate induction, but there was no significant difference compared to the control group. In the S+ group, although EGFP was expressed, the fluorescence was weak and the expression level was low.</p>
<p>We speculate that this may be due to compatibility issues after the tetrathionate sensor TtrSR was transferred from E. coli to Saccharomyces cerevisiae. Due to differences in protein expression and delivery systems between eukaryotic and prokaryotic cells, TtrS may not effectively realize its function in Saccharomyces cerevisiae due to issues such as signal peptides and transmembrane domains, and may not be properly localized to the cell membrane. Additionally, the expression levels of TtrS and TtrR may be insufficient in brewing yeast. In subsequent experiments, we plan to optimize TtrS and TtrR, including codon optimization, signal peptide prediction and optimization, membrane localization prediction, and transmembrane domain optimization, in order to enable TtrRS to function better in brewing yeast.</p>
<p>We speculate that this may be due to compatibility issues after the tetrathionate sensor TtrSR was transferred from E. coli to <i>Saccharomyces cerevisiae</i>. Due to differences in protein expression and delivery systems between eukaryotic and prokaryotic cells, TtrS may not effectively realize its function in <i>Saccharomyces cerevisiae</i> due to issues such as signal peptides and transmembrane domains, and may not be properly localized to the cell membrane. Additionally, the expression levels of TtrS and TtrR may be insufficient in brewing yeast. In subsequent experiments, we plan to optimize TtrS and TtrR, including codon optimization, signal peptide prediction and optimization, membrane localization prediction, and transmembrane domain optimization, in order to enable TtrRS to function better in brewing yeast.</p>
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<h3>Big circle2: Adhesion protein</h3>
<p>To enable Saccharomyces cerevisiae to “sense” the presence of IBD and colonize the corresponding sites, we utilize an adhesion molecule for this function. Through literature research, we have found that the adhesion protein Als3 from Candida albicans can bind to E-cadherin on epithelial cells, thereby achieving adhesion. Since Saccharomyces cerevisiae and Candida albicans are both common fungi in the human body and share some similarities, we have decided to express Als3 in Saccharomyces cerevisiae to achieve adhesion to intestinal epithelial cells.</p>
<p>To enable <i>Saccharomyces cerevisiae</i> to “sense” the presence of IBD and colonize the corresponding sites, we utilize an adhesion molecule for this function. Through literature research, we have found that the adhesion protein Als3 from Candida albicans can bind to E-cadherin on epithelial cells, thereby achieving adhesion. Since <i>Saccharomyces cerevisiae</i> and Candida albicans are both common fungi in the human body and share some similarities, we have decided to express Als3 in <i>Saccharomyces cerevisiae</i> to achieve adhesion to intestinal epithelial cells.</p>
<p>We conducted an adhesion ability assay using transformed Saccharomyces cerevisiae on small intestinal tissue. The final results indicate that after expressing Als3 in yeast cells, the adhesion ability of these cells to small intestinal tissue has improved to some extent. However, there is no significant advantage compared to the WT (wild-type) cells.</p>
<p>We conducted an adhesion ability assay using transformed <i>Saccharomyces cerevisiae<i> on small intestinal tissue. The final results indicate that after expressing Als3 in yeast cells, the adhesion ability of these cells to small intestinal tissue has improved to some extent. However, there is no significant advantage compared to the WT (wild-type) cells.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 16 <b>Statistical results of quantification of attachment experiment</b> (Als3: the attachment of saccharomyces cerevisiae expressing Als3; WT: the attachment of wild-type saccharomyces cerevisiae, control groups) </p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 16 <b>Statistical results of quantification of attachment experiment</b> (Als3: the attachment of <i>saccharomyces cerevisiae</i> expressing Als3; WT: the attachment of wild-type <i>saccharomyces cerevisiae</i>, control groups) </p>
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<h5>Learn</h5>
<p>The experimental results indicate that expressing Als3 can enhance adhesion ability to some extent. In order to explore better solutions, we need to test the adhesion capabilities of more adhesion molecules or systems. We plan to introduce CSP or PfEMP1 from Plasmodium, surface glycoproteins from Giardia lamblia, or surface antigen SAG1 into Saccharomyces cerevisiae , and compare their adhesion effects after expression in Saccharomyces cerevisiae.</p>
<p>The experimental results indicate that expressing Als3 can enhance adhesion ability to some extent. In order to explore better solutions, we need to test the adhesion capabilities of more adhesion molecules or systems. We plan to introduce CSP or PfEMP1 from Plasmodium, surface glycoproteins from Giardia lamblia, or surface antigen SAG1 into <i>Saccharomyces cerevisiae</i> , and compare their adhesion effects after expression in <i>Saccharomyces cerevisiae</i>.</p>
