<p>To identify the lesion site, we need a molecule that specifically characterizes IBD. Current research suggests that thiosulfate and tetrathionate can serve as indicators of intestinal inflammation <sup>1</sup>, and the levels of thiosulfate or tetrathionate are directly proportional to the severity of intestinal inflammation. A previous study constructed a tetrathionate sensor in E. coli <sup>2</sup>. This system is the TtrSR two-component system (TCS) from the marine bacterium Shewanella halifaxensis HAW-EB4. The TCS includes TtrS, 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>
<p>To verify the effectiveness of the tetrathionate sensor TtrSR, we designed the corresponding plasmids. We planed to express EGFP downstream of ttrB and validate the effect of TtrSR by testing the fluorescence intensity of the cells. We used Ura-HIS nutrient-deficient medium to select yeast that had been successfully transformed. Then, we tested the effectiveness of the TtrSR system using confocal microscopy. For more details, please refer to protocol.</p>
<p>Aim:</p>
<p><b>Aim:</b></p>
<p>To confirm the effectiveness of TtrRS in <i>Saccharomyces cerevisiae</i></p>
<p>Agglutinin-like sequence protein3 (Als3) is a cell surface glycoprotein of Candida albicans that plays a crucial role in vitro adhesion and biofilm formation. Als3 is essential for the binding of Candida albicans hyphae to various host cell surface receptor proteins, and it induces endocytosis by binding to E-cadherin on epithelial cells <sup>3</sup>. Since our chassis organism, <i>Saccharomyces cerevisiae</i>, is also a fungus like Candida albicans, expressing Als3 on the surface of <i>Saccharomyces cerevisiae</i> cells is expected to enable binding to E-cadherin on the small intestinal epithelium, thereby allowing the engineered bacteria to colonize the small intestine.</p>
<p>To verify the effectiveness of Als3, we designed an adhesion assay experiment.<sup>4</sup> We designed the corresponding plasmids to express Als3 in <i>Saccharomyces cerevisiae</i> and simultaneously used the expression of EGFP for yeast tracking. After transforming the plasmids into <i>Saccharomyces cerevisiae</i>, we selected yeast that had been successfully transformed using Ura nutrient-deficient medium. Then, we verified the effectiveness of Als3 through the adhesion capability assay. For further details, please refer to the protocol</p>
<p>Aim:</p>
<p><b>Aim:</b></p>
<p>To verify the change in adhesion capability of <i>Saccharomyces cerevisiae</i> expressing Als3 to small intestinal cells </p>
