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<p>This system is the TtrSR two-component system (TCS) from the marine bacterium Shewanella halifaxensis HAW-EB4. The TCS consists of TtrS and TtrR. TtrS is a membrane-bound sensor histidine kinase (SK), which can phosphorylate the cytoplasmic response regulator (RR) TtrR in the presence of tetrathionate. Phosphorylated TtrR activates the expression of downstream genes through the ttrB promoter (PttrB).</p>
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<h2 id="Experiment">
<h2>Experiment</h2>
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<p>To test the effectiveness of the tetrathionate sensor TtrSR, we designed the corresponding plasmids (Fig.3 ttrR, Fig.4 ttrS). We arranged for EGFP to be expressed downstream of ttrB and validated the effectiveness of TtrSR by measuring the fluorescence intensity of the cells. After successfully transforming the plasmids into Saccharomyces cerevisiae, we selected three well-growing colonies for amplification. （Fig. after transforming）Then, we divided the cells from the same clone into two groups, one group induced with 1mM tetrathionate and the other group without inducer as control group. After 12 hours of induction, an appropriate amount of bacterial solution was prepared into temporary slides and the fluorescence intensity was tested using a fluorescence confocal microscope. For more details, please refer to the protocol.</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 3 ttrR</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/ alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 4 ttrS</p>
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<p>To test the effectiveness of Als3, we designed a bacterial attachment experiment. We constructed the corresponding plasmid and after transforming it into Saccharomyces cerevisiae, we used human intestinal sections to test the adhesion effects and differences between S. cerevisiae expressing Als3 and yeast without Als3 expression. For more details, please refer to the protocol.</p>
<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 5 Als3</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/ alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 6 转化后</p>
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<h2 id="Result">
<h2>Result</h2>
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<h3>Tetrathionate sensor TtrSR</h3>
<p>After acquiring the fluorescence confocal microscopy images, we calculated the ratio of total fluorescence intensity to the number of cells.</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 7 Confocal images of one group with induction and one without</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 8 Statistical graph</p>
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<p>The results indicate that after induction, the expression of the downstream protein increased, but there was no significant difference compared to the control group as anticipated.</p>
<p>This could be due to compatibility issues that arise when the tetrathionate sensor TtrSR is transferred from E. coli to Saccharomyces cerevisiae. Because of differences between eukaryotic and prokaryotic protein expression and delivery systems, TtrS may not be properly processed and localized to the cell membrane in S. cerevisiae. In subsequent experiments, we optimized the TtrS protein, including codon optimization, structural modeling and prediction (still being modified——, to be added later).</p>
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<h3>Adhesive protein Als3</h3>
<p>After conducting the attachment experiment, we counted the yeast cells left on the tissue and calculated the ratio of the number of cells to the tissue area.</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 9 Tissue staining image</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 8 Light microscopy image</p>
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<div class="image-container" style="display: flex; flex-direction: column; align-items: center;">
<img src="https://static.igem.wiki/teams/5187/wiki-colonization-fig/" alt="ibd_figure" class="shadowed-image" style="width: 90%; max-width: 800px;">
<p style="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 10 Statistical graph</p>
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<p>The results indicate that after expressing Als3, the adhesion ability of Saccharomyces cerevisiae to intestinal tissue has significantly improved. It has been demonstrated that Als3 can indeed be used for colonization in Saccharomyces cerevisiae.</p>
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<h2 id="Protocol">Protocol</h2>
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<p>
<a href="https://static.igem.wiki/teams/5187/wiki-colonization-fig/saccharomyces-cerevisiae-sensor-system-induced-expression-protocol.pdf" target="_blank" rel="noopener noreferrer">
Saccharomyces cerevisiae Sensor System-Induced Expression (click me to open the protocol!)
</a>
</p>
<p>
<a href="https://static.igem.wiki/teams/5187/wiki-colonization-fig/tissue-binding-assay-protocol.pdf" target="_blank" rel="noopener noreferrer">
Tissue binding assay (click me to open the protocol!)
</a>
</p>
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