<p>For more information, please refer to <ahref="https://2024.igem.wiki/tsinghua/therapy-system"style="color: #FF5151">Therapy system</a>.</p>
<h4>Communication with relevant social stakeholders</h4>
<p>To assess the potential for further applications of the muscone molecular switch in <i>Saccharomyces cerevisiae</i>, we communicated with Bluepha Company, which specializes in developing new microbial fermentation materials through synthetic biology. Bluepha Company's feedback indicated that the muscone molecular switch in our modified <i>Saccharomyces cerevisiae</i> could serve as an alternative to the traditional methanol promoter used in Pichia pastoris fermentation systems. Muscone offers cost-effectiveness and higher safety compared to methanol, making it an attractive option for the design of innovative fermentation processes in <i>Saccharomyces cerevisiae</i>.</p>
<p>Shortcoming: Background signal noise still exists; There are significant differences in baseline expression between different strains.</p>
<p>Solution: Take further measures to modify the yeast genome to eliminate background noise; Introduce the musk ketone molecular switch gene into the yeast genome to avoid the impact of plasmid cloning variations, and screen for superior yeast strains for cultivation.</p>
<p><b>Shortcoming</b>: Background signal noise still exists; There are significant differences in baseline expression between different strains.</p>
<p><b>Solution</b>: Take further measures to modify the yeast genome to eliminate background noise; Introduce the musk ketone molecular switch gene into the yeast genome to avoid the impact of plasmid cloning variations, and screen for superior yeast strains for cultivation.</p>
<h3>Proof of secretion of lactate</h3>
<h4>Previous literature research</h4>
<p>We selected a small molecule drug that is relatively easy to biosynthesize and can be readily secreted to treat diseases by passing through intestinal wall cells. Through literature search, we found that Liliana M Sanmarco et. discovered a signaling pathway in which lactic acid inhibits the abnormal activation of immune cells<sup>[3]</sup>. In their study, they designed an engineered E. coli bacteria to secrete lactic acid and successfully suppressed dendritic cells and T cells in the intestinal tract. This provided a reference for our choice. We decided to introduce lactate dehydrogenase into brewing yeast, altering the anaerobic metabolism pathway to synthesize D-lactic acid for therapeutic purposes.</p>
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<p>We introduced a lactate dehydrogenase gene expressed by the galactose promoter into <i>Saccharomyces cerevisiae</i> and measured the content of D-lactic acid in the supernatant after induction. This validated that by introducing lactate dehydrogenase, we can alter the anaerobic metabolic pathway of S. cerevisiae to synthesize D-lactic acid, and the produced D-lactic acid can be secreted into the surrounding environment of the yeast.</p>
<p>For more information, please refer to <ahref="https://2024.igem.wiki/Tsinghua/description"style="color: #FF5151">Description</a>.</p>
<p>Shortcoming: Experimental findings have shown that the nutritional composition of the yeast culture environment has a significant impact on the secretion of lactic acid; In the anaerobic environment, yeast can still produce alcohol, which may have an impact on the health of patients.</p>
<p>Solution:By further modifying the yeast's anaerobic metabolic pathways through genomic engineering, the impact of the nutritional composition of the culture environment on lactic acid secretion can be reduced; knockout of the alcohol dehydrogenase gene in the yeast genome.</p>
<p><b>Solution</b>:By further modifying the yeast's anaerobic metabolic pathways through genomic engineering, the impact of the nutritional composition of the culture environment on lactic acid secretion can be reduced; knockout of the alcohol dehydrogenase gene in the yeast genome.</p>
<p>We tested the signal reporting intensity of the TtrSR two-component system introduced into <i>Saccharomyces cerevisiae</i> under the induction condition of tetrathionate presence using the GFP reporter gene. The results showed that the signal reporting intensity was higher than that of the control group, but not significantly. We have discussed the experimental results in detail in the Colonization system section.</p>
<h4>Communication with relevant social stakeholders</h4>
<p>To evaluate whether the signal biomarker for IBD that we used has diagnostic value, we conducted an interview with Dr. Li Yue from Peking Union Medical College Hospital. Dr. Li Yue specializes in the diagnosis and treatment of intestinal diseases and has extensive experience in the clinical diagnosis and treatment of IBD. Dr. Li Yue pointed out that, as an adjunct to treatment, using tetrathionate as a signal biomarker for IBD has certain diagnostic value, but in clinical treatment, more indicators are needed to judge the condition of the disease.</p>
<p>Shortcoming: The signal biomarkers cannot fully represent the condition of the disease; The binary component system is not highly sensitive in eukaryotic systems.</p>
<p>Solution: Using muscone as a drug secretion switch for auxiliary treatment, and employing the apoptosis system to control biological safety; by referring to the research of the team, operations such as eukaryotic codon optimization have been performed on the two-component system to make it compatible with eukaryotic systems. For details, please see the Colonization system.</p>
<p><b>Shortcoming</b>: The signal biomarkers cannot fully represent the condition of the disease; The binary component system is not highly sensitive in eukaryotic systems.</p>
<p><b>Solution</b>: Using muscone as a drug secretion switch for auxiliary treatment, and employing the apoptosis system to control biological safety; by referring to the research of the team, operations such as eukaryotic codon optimization have been performed on the two-component system to make it compatible with eukaryotic systems. For details, please see the Colonization system.</p>
<h3>Proof of colonization protein</h3>
<h4>Previous literature research</h4>
<p>By referring to the research of Wang Tianming et al.<sup>[6]</sup>, we selected agglutinin-like sequence protein 3 (Als3) as the adhesion protein for <i>Saccharomyces cerevisiae</i> to adhere to the intestinal wall cells. This protein originates from Candida albicans and binds to epithelial E-cadherin. Due to its close phylogenetic relationship with <i>Saccharomyces cerevisiae</i>, we anticipate that it can produce the same adhesion effect in <i>Saccharomyces cerevisiae</i> as it does in Candida albicans.</p>
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</div>
<p>We determined the location of <i>Saccharomyces cerevisiae</i> by expressing GFP in the yeast and examined the colonization of <i>Saccharomyces cerevisiae</i> after co-incubation with small intestinal sections. The results confirmed that the expression of Als3 in <i>Saccharomyces cerevisiae</i> achieved colonization on small intestinal epithelial cells.</p>
<p>For more information, please refer to <ahref="https://2024.igem.wiki/tsinghua/colonization"style="color: #FF5151">Colonization system</a>.</p>
<p>Shortcoming: There is insufficient experimental evidence to verify the colonization effect; No complete experimental construction and testing of the colonization system have been conducted.</p>
<p>Solution: We will verify the expression of Als3 on the membrane of <i>Saccharomyces cerevisiae</i> using techniques such as Western blot; Attempt to construct and test a complete colonization system experimentally.</p>
<p><b>Shortcoming</b>: There is insufficient experimental evidence to verify the colonization effect; No complete experimental construction and testing of the colonization system have been conducted.</p>
<p><b>Solution</b>: We will verify the expression of Als3 on the membrane of <i>Saccharomyces cerevisiae</i> using techniques such as Western blot; Attempt to construct and test a complete colonization system experimentally.</p>
