<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 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 (Levitt et al., 1999), 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 (Kristina N-M Daeffler, 2017). 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>To confirm the effectiveness of TtrRS in Saccharomyces cerevisiae</p>
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<h3>Adhesion Protein Als3</h3>
<p>We used human small intestinal sections to test the adhesion effects and differences between Saccharomyces cerevisiae expressing Als3 and yeast without Als3 expression. The yeast expressing Als3 was transformed with the pYES2-SV40-ALS3-EGFP plasmid, while the yeast without Als3 expression was also transformed with plasmid that could express EGFP, allowing for the observation of Saccharomyces cerevisiae. The human small intestinal sections were purchased from Zhongke Guanghua Company. After the adhesion assay, we stained the sections with Hoechst 33342. This staining allowed the small intestinal cells to emit blue fluorescence. Therefore, we could observe the small intestinal tissue through the blue fluorescence, while the green fluorescence marked the position of Saccharomyces cerevisiae. We counted the yeast cells adhered to the tissue and calculated the ratio of cell number to tissue area. The images were processed using Image J, and statistical graphs were plotted using GraphPad Prism.</p>
