<p>The engineered bacteria we designed will colonize the patient's intestinal tract, achieving long-term regulated secretion for drug delivery, enhancing the stability of treatment and reducing costs. The validation of the colonization system will be conducted from two aspects: the perception of IBD signals and the colonization proteins, verifying both the principles and the results.</p>
<h3>Proof of IBD marker sensor</h3>
<h4>Previous literature research</h4>
<p>Out of consideration for patient safety and therapeutic efficacy, we aim to specifically colonize the engineered bacteria on the intestinal segments of the patient's lesion area. Through discussions with members of the Tsinghua 2021 team, we have selected tetrathionate as the signaling biomarker for intestinal inflammation<suo>[4]</suo>.</p>
<p>Referencing the research conducted by Kristina and colleagues<sup>[5]</sup>, we have incorporated the TtrSR two-component system (TCS) from marine bacteria into Saccharomyces cerevisiae to function as a sensing system for detecting signals characteristic of IBD (Inflammatory Bowel Disease).</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 5 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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<p>We tested the signal reporting intensity of the TtrSR two-component system introduced into Saccharomyces cerevisiae 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>
<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 Saccharomyces cerevisiae 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 Saccharomyces cerevisiae, we anticipate that it can produce the same adhesion effect in Saccharomyces cerevisiae as it does in Candida albicans.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 7 Statistical results of quantification of attachment experiment (Als3: the attachment of saccharomyces cerevisiae expressing Als3; WT: the attachment of wild-type saccharomyces cerevisiae, control groups)</p>
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<p>We determined the location of Saccharomyces cerevisiae by expressing GFP in the yeast and examined the colonization of Saccharomyces cerevisiae after co-incubation with small intestinal sections. The results confirmed that the expression of Als3 in Saccharomyces cerevisiae 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 Saccharomyces cerevisiae using techniques such as Western blot; Attempt to construct and test a complete colonization system experimentally.</p>
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<h2>Safety</h2>
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<h3>Proof of apoptosis</h3>
<h4>Communication with relevant social stakeholders</h4>
<p>We discussed the design of a biosafety suicide system for intestinal environmental markers with Dr. Liu Zhihua, and Professor Liu recommended that we use bile acids as markers for the intestinal environment.</p>
<h4>Previous literature research</h4>
<p>We selected bile acids as the marker for the intestinal environment, based on previous studies using the bile acid receptor FXR and its downstream BSEP promoter as the sensing system for the marker<sup>[7]</sup>, and employed the Ci protein from the λ phage as an inhibitory element to suppress the expression of the toxin protein MazF.</p>
<p>For more information please refer to <ahref="https://2024.igem.wiki/tsinghua/safety"style="color: #FF5151">Safety</a>.</p>
<h3>Proof of chassis creatures</h3>
<h4>Communication with relevant social stakeholders</h4>
<p>We discussed using Saccharomyces cerevisiae as the chassis organism for our project with Dr. Liu Zhihua. Professor Liu affirmed our design of using Saccharomyces cerevisiae as the chassis organism and pointed out that Saccharomyces cerevisiae is distributed in the human intestinal environment, being a relatively harmless and safe microorganism to humans, making it very suitable as the chassis organism for our project.</p>
<p>We discussed the type of engineered strain of Saccharomyces cerevisiae with Dr. Li Peng. Professor Li recommended that we use the MAT alpha genotype strain, which has nutritional deficiencies in his, leu, lys, and ura. This strain can be used for transformation and screening during the project's experimental process and prevent safety threats from biological leakage.</p>
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<h2>Refernece</h2>
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<p>[1] Wu X, Yu Y, Wang M, Dai D, Yin J, Liu W, Kong D, Tang S, Meng M, Gao T, Zhang Y, Zhou Y, Guan N, Zhao S, Ye H. AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation. Nat Commun. 2024 Feb 6;15(1):1122. doi: 10.1038/s41467-024-45383-z. PMID: 38321056; PMCID: PMC10847102.</p>
<p>[2] Scott BM, Gutiérrez-Vázquez C, Sanmarco LM, da Silva Pereira JA, Li Z, Plasencia A, Hewson P, Cox LM, O'Brien M, Chen SK, Moraes-Vieira PM, Chang BSW, Peisajovich SG, Quintana FJ. Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nat Med. 2021 Jul;27(7):1212-1222. doi: 10.1038/s41591-021-01390-x. Epub 2021 Jun 28. PMID: 34183837.</p>
<p>[3] Sanmarco LM, Rone JM, Polonio CM, Fernandez Lahore G, Giovannoni F, Ferrara K, Gutierrez-Vazquez C, Li N, Sokolovska A, Plasencia A, Faust Akl C, Nanda P, Heck ES, Li Z, Lee HG, Chao CC, Rejano-Gordillo CM, Fonseca-Castro PH, Illouz T, Linnerbauer M, Kenison JE, Barilla RM, Farrenkopf D, Stevens NA, Piester G, Chung EN, Dailey L, Kuchroo VK, Hava D, Wheeler MA, Clish C, Nowarski R, Balsa E, Lora JM, Quintana FJ. Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells. Nature. 2023 Aug;620(7975):881-889. doi: 10.1038/s41586-023-06409-6. Epub 2023 Aug 9. PMID: 37558878; PMCID: PMC10725186.</p>
<p>[4] Levitt MD, Furne J, Springfield J, Suarez F, DeMaster E. Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa. J Clin Invest. 1999 Oct;104(8):1107-14. doi: 10.1172/JCI7712. PMID: 10525049; PMCID: PMC408582.</p>
<p>[6] Wächtler B, Citiulo F, Jablonowski N, Förster S, Dalle F, Schaller M, Wilson D, Hube B. Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process. PLoS One. 2012;7(5):e36952. doi: 10.1371/journal.pone.0036952. Epub 2012 May 14. PMID: 22606314; PMCID: PMC3351431.</p>
<p>[7] Jiang L, Zhang H, Xiao D, Wei H, Chen Y. Farnesoid X receptor (FXR): Structures and ligands. Comput Struct Biotechnol J. 2021 Apr 20;19:2148-2159. doi: 10.1016/j.csbj.2021.04.029. Erratum in: Comput Struct Biotechnol J. 2022 Mar 01;20:1227-1228. doi: 10.1016/j.csbj.2022.02.029. PMID: 33995909; PMCID: PMC8091178.</p>
<p>We chose the mating pathway in Saccharomyces cerevisiae as the conduit for muscone signaling in yeast. Using the mating pathway’s pFUS1 promoter, we expressed the downstream lactate dehydrogenase to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae, secreting lactic acid for the treatment of IBD. Initially, we designed a plasmid with the pFUS1 promoter expressing the GFP reporter gene and screened the successfully transformed yeast using Ura nutritional deficiency. We then tested the effectiveness of the muscone molecular switch using confocal microscopy; for details, please refer to the protocol. Subsequently, we designed the pFUS1 promoter to express lactate dehydrogenase from E. coli. By co-transforming it with Muscone Receptor & Gα (pESC) into Saccharomyces cerevisiae, we achieved the construction of the complete pathway.</p>
