<p>To better construct a subsystem that allows Saccharomyces cerevisiae to colonize IBD lesions, we divided it into two sub-parts: one part is the IBD signaling molecule receptor, and the other part is the adhesion protein. Only after confirming that both parts are functioning properly will we combine these two sub-parts. This section is divided into two main cycles.</p>
<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>
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<h3>Big circle1: Tetrathionate sensor</h3>
<p>To enable Saccharomyces cerevisiae to “sense” the presence of IBD, we need a receptor for IBD.</p>
<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>
<h4>Cycle1</h4>
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<h5>Design</h5>
<p>After conducting a literature review, we decided to introduce the tetrathionate sensor TtrSR into yeast cells to serve as a receptor for IBD.</p>
<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>
<h5>Build</h5>
<p>After confirming the sequence, we designed plasmids that can construct the tetrathionate sensor TtrSR system within Saccharomyces cerevisiae, each carrying the URA3 and HIS3 genes as selection marers, as shown in the figure below.</p>
<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>
<p>We induced the transformed yeast with 1mM K<sub>2</sub>O<sub>6</sub> S<sub>4</sub> or without K<sub>2</sub>O<sub>6</sub> S<sub>4</sub> (as a control) and measured the expression level of EGFP after 12 hours of induction. The final results showed that after constructing the tetrathionate sensor TtrSR system in yeast cells, the cells were able to sense the IBD signal tetrathionate and activate the expression of downstream genes. However, compared to the control group, there was no significant increase in the expression level of the downstream protein. Although the downstream protein was expressed in response to the IBD signal, the expression level still needs to be improved.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 14 Tetrathionate induction experiment (s: control group without inducer; s+: K<sub>2</sub>O<sub>6</sub> S<sub>4</sub> added)</p>
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<p>We induced the transformed yeast with 1 mM K<sub>2</sub>O<sub>6</sub>S<sub>4</sub> or without K<sub>2</sub>O<sub>6</sub>S<sub>4</sub> (control) and measured the expression of EGFP at 0.5, 1, 2, 4, 8, 12, and 24 hours post-induction. However, the final results from the fluorescence confocal microscopy showed no fluorescence in either the experimental or control groups.</p>
<h5>Learn</h5>
<p>Regarding this phenomenon, after discussion, we hypothesize that it may be due to the low expression levels of TtrS and TtrR, or it could be that the membrane protein TtrS, originally from prokaryotic cells, is unable to effectively localize and anchor to the membrane after being expressed in yeast cells. We need to further optimize the relevant sequences.</p>
<p>Regarding this phenomenon, we speculate that it might be due to the fact that the samples were not immediately prepared and observed after being collected at different time points. Instead, they were all collected first and then prepared for observation after some time had passed, which could have led to cell death, protein degradation, or fluorescence quenching. It is also possible that the induction conditions were not suitable. Therefore, we need to modify the protocol and experimental plan.</p>
<h4>Cycle2</h4>
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<h5>Design</h5>
<p>To address the issue of insufficient downstream protein expression, we decided to optimize the relevant sequences to improve the efficiency of the tetrathionate sensor TtrSR system.</p>
<p>To address the issue of not being able to observe fluorescence, we have made revisions to the existing experimental protocol to eliminate interference from unrelated factors on the experimental results. </p>
<h5>Build</h5>
<p>After reviewing relevant literature, we further optimized the codons of TtrS and TtrR to enable more efficient expression in yeast cells. Additionally, we optimized TtrS by adding signal peptides and improving the transmembrane sequences.</p>
<p>We conducted predictions for the optimized TtrS, including signal peptide cleavage predictions and intracellular membrane localization predictions.</p>
<p>We increased the concentration of the tetrathionate inducer from 1mM to 2mM and collected the samples immediately after 24 hours of induction to prepare temporary slides for observation.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 14 Statistical results of tetrathionate induction experiment (s: control group without inducer; s+: K<sub>2</sub>O<sub>6</sub>S<sub>4</sub> added)</p>
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<h5>Learn</h5>
<p>After optimization, the localization and anchoring of TtrS showed some improvement. To further test the system, we need to conduct additional tests on the inducer concentration and induction time.</p>
<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>
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<h3>Big circle2: Adhesion protein</h3>
<p>To enable Saccharomyces cerevisiae to colonize the corresponding site after "sensing" the presence of IBD, we used an adhesion protein to achieve this function.</p>
<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>
<h4>Cycle1</h4>
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<h5>Design</h5>
<p>After conducting a literature review, we decided to express the adhesion protein Als3 from Candida albicans in yeast cells to achieve adhesion to intestinal epithelial cells.</p>
<p>We have decided to express both Als3 and EGFP in yeast cells and to test the function of Als3 in brewing yeast through adhesion assay experiments.</p>
<h5>Build</h5>
<p>After conducting a literature review, we decided to express the adhesion protein Als3 from *Candida albicans* in yeast cells to achieve adhesion to intestinal epithelial cells.</p>
<p>After confirming the sequence, we designed a plasmid that can express Als3 in brewing yeast, using EGFP as a tracing marker. Additionally, this plasmid carries URA3 as a selection marker. Figure below shows the details.</p>
<p>We conducted an adhesion assay experiment to assess the ability of the transformed yeast to adhere to colon tissue. The final results showed that after expressing Als3 in yeast cells, their adhesion ability to colon tissue significantly improved. However, upon observing the fluorescence expression in Saccharomyces cerevisiae, we found that the fluorescence expression was relatively weak, with many cells not exhibiting strong fluorescence.</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>
<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>
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<h5>Learn</h5>
<p>Regarding this phenomenon, upon further observation of the relevant slides, we found that *Saccharomyces cerevisiae* on the tissue could be effectively distinguished and counted under an optical microscope. To more accurately characterize the adhesion ability of Saccharomyces cerevisiae, we need to further optimize the observation methods.</p>
<h4>Cycle2</h4>
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<h5>Design</h5>
<p>Based on the observations under the optical microscope, we plan to repeat the adhesion assay experiment while improving the observation and counting methods for Saccharomyces cerevisiae.</p>
<h5>Build</h5>
<p>We will continue using the same yeast strains and procedures as before but modify the observation techniques.</p>
<h5>Test</h5>
<p>We re-conducted the counting and analysis, with the relevant results shown in the figure below. As can be seen, we were still able to demonstrate that after expressing Als3, the colonization ability of Saccharomyces cerevisiae showed a significant improvement.</p>
<p>With the improved observation techniques, we can now more accurately characterize the adhesion ability of *Saccharomyces cerevisiae*. Moving forward, we can perform in vivo experiments or use IBD-affected tissues to assess the adhesion ability, allowing for a more comprehensive evaluation of the adhesion capacity of the modified 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 Saccharomyces cerevisiae , and compare their adhesion effects after expression in Saccharomyces cerevisiae.</p>
