<li><ahref="#Inhalation of muscone">Inhalation of muscone</a></li>
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<h2id="description">
<h2>General Description</h2>
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<p>Inflammatory Bowel Disease (IBD) is an indeterminate colitis and autoimmune inflammatory bowel disease that involves the ileum, rectum, and colon. Due to its unknown pathogenesis, it is difficult to cure and prone to relapse, which is then referred to as the “green cancer”. As the incidence of IBD is increasing globally, the poor therapeutic experience, which the patients are required to receive long-term hormone injection and biologic combination therapy, is determined to seek a breakthrough. Our team stands to develop a more favorable treatment experience. Relevant studies have shown that lactic acid has an inhibitory effect on abnormally activated dendritic cells and cytotoxic T-cells in autoimmune diseases, and thus is expected to improve the symptoms of IBD patients. Based on the research, we designed a yeast engineered bacterium. We introduced a muscone detecting switch in the engineered yeast and activated the downstream pathway to express lactate dehydrogenase to produce lactic acid. Ultimately, the engineered bacteria will colonize the small intestine, enabling the patient to treat IBD by inhaling muscone gas in vitro therefore induces the yeast to synthesize a low amount of stabilized lactic acid in vivo. Aroma therapy has the potential to be a new trend in long-term treatment for IBD as it can reduce the discomfort of treatment and improve the long-term treatment experience. </p>
<h2id="Introduction">
<h2>Introduction</h2>
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<p>The core of our treatment project revolves around three aspects: </p>
<p>Effectiveness: The therapy system we designed can effectively induce yeast to secrete lactic acid; </p>
<p>Safety: Our engineered yeast can specifically colonize the patient's lesion site and die after leaving the patient's body environment; </p>
<p>Feasibility: Muscone can reach the intestines at a higher concentration through inhalation.</p>
<p>We designed detailed physical and chemical experiments to verify the first two aspects; for the third point, due to the safety restrictions imposed by iGEM on participating teams, we only used theoretical models such as gas molecule diffusion to illustrate it. Unfortunately, the plasmid expression system in yeast is not very stable. In addition, the molecular switch of muscone we designed has a complex interplay with the existing signaling pathways in yeast cells.</p>
<p>Therefore, validating experiments is very difficult, especially considering the time constraints of the iGEM competition. Despite referencing a large amount of papers and materials, most of the data we obtained is still frustrating. It is also very difficult to explain these anomalous data. We have overcome all these obstacles and successfully established a more mature muscone molecular switch and colonization system in the yeast system. This indicates that the concept of inhalation therapy with muscone is a theoretically feasible one. </p>
<p>The muscone molecular switch we introduced from the yeast system is a very novel, simple, and responsive regulatory method. By modifying the downstream response signals, it can be easily applied to the design of other genetic engineering projects.</p>
<p>We introduced the lactate dehydrogenase gene (ldhA) regulated by a galactose promoter into yeast cells, induced it under different conditions, and measured the lactic acid content in the supernatant. We found that, aside from a small amount of leakage expression of the ldhA gene itself, galactose can very efficiently induce yeast to secrete sufficient lactic acid into the external environment. It is worth noting that because glucose is a better carbon source for yeast compared to galactose, the difference in carbon sources can have some effects on lactic acid secretion beyond just gene expression, which we have not demonstrated here. There is a more detailed discussion in the wet lab section.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 2 Results of yeast-induced lactic acid secretion. This image shows the differences in lactic acid secretion by yeast induced by different carbon sources (glucose or galactose).(wt: wild-type yeast, ldhA: yeast transformed with a plasmid containing the ldhA gene)</p>
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<h2>Topic1</h2>
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<p>Tsinghua University is a public university in Haidian, Beijing. It is affiliated with and funded by the Ministry of Education of China. The university is part of Project 211, Project 985, and the Double First-Class Construction. It is also a member of the C9 League. Tsinghua University's campus is situated in northwest Beijing, on the site of the former imperial gardens of the Qing dynasty. Currently, the university has 21 schools and 59 departments, with faculties in science, engineering, humanities, law, medicine, history, philosophy, economics, management, education and art.</p>
<p>Notable alumni who have held senior positions in Chinese politics include current general secretary and president of China, Xi Jinping, former general secretary and president of China Hu Jintao, former chairman of the National People's Congress Wu Bangguo, former premier Zhu Rongji, and the former first vice premier Huang Ju. This also includes politicians like Wu Guanzheng, former governor of the People's Bank of China Zhou Xiaochuan, former minister of finance Lou Jiwei, general Sun Li-jen, Liang Qichao, and more. Since 2016, Tsinghua graduates who have political prominence are disproportionately greater in number than graduates of other famous universities.</p>
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<h3>The muscone molecular switch can function efficiently</h3>
<p>We simultaneously introduced plasmid vectors expressing the muscone receptor and the corresponding Gα protein into yeast cells, along with a plasmid vector expressing the GFP protein controlled by the pFUS1 promoter. We used galactose to induce the expression of receptors and Gα proteins, and added a certain concentration of muscone for induction. The results are as follows. We found that, aside from the weak background expression, muscone can very efficiently induce the expression of the downstream controlled GFP gene.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 3 Yeast specifically expresses GFP through designed pathways in the condition of muscone inducing</p>
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<h2>Topic2</h2>
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<h3>The muscone molecular switch can control lactic acid secretion in a concentration-dependent manner</h3>
<p>We combined the muscone molecular switch with the lactate dehydrogenase gene to measure the differences in the rate of lactic acid secretion by yeast induced by different concentrations of muscone.</p>
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<h2id="Colonization system">
<h2>Colonization system</h2>
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<p>Tsinghua University engages in extensive research and offers 51 bachelor's degree programs, 139 master's degree programs, and 107 doctoral programs through 20 colleges and 57 departments covering a broad range of subjects, including science, engineering, arts and literature, social sciences, law, medicine. Along with its membership in the C9 League, Tsinghua University affiliations include the Association of Pacific Rim Universities, a group of 50 leading Asian and American universities, Washington University in St. Louis's McDonnell International Scholars Academy, a group of 35 premier global universities, and the Association of East Asian Research Universities, a 17-member research collaboration network of top regional institutions. Tsinghua is an associate member of the Consortium Linking Universities of Science and Technology for Education and Research (CLUSTER). Tsinghua is a member of a Low Carbon Energy University Alliance (LCEUA), together with the University of Cambridge and the Massachusetts Institute of Technology (MIT).</pp>
<p>School of Life Sciences was first established in 1926 under the name Department of Biology. Botanist Qian Chongshu took up the first dean.During the nationwide reorganization of universities in the early 1950s, the Department of Biology was merged into other universities, namely Peking University etc., resulting in a vacancy in the field of biological research in Tsinghua for almost 30 years.In June 1984, decisions were made about the reestablishment of the Department of Biology, and the department officially reopened in September. During the reestablishment the Department of Biology of Peking University, the Institute of Biophysics of Chinese Academy of Sciences, and many other institutes as well as biologists provided valuable support and help. The department changed its name to the current name in September 2009. As of 2013, structural biologist and foreign associate of National Academy of Sciences of United States Dr. Wang Hongwei (王宏伟) is the current dean of School of Life Sciences. The school currently has 129 professors and employees, around 600 undergraduates (including the candidates of Tsinghua University – Peking Union Medical College joint MD program).</p>
<p>Admission to Tsinghua for both undergraduate and graduate schools is extremely competitive. Undergraduate admissions for domestic students is decided through the gaokao, the Chinese national college entrance exam, which allows students to list Tsinghua University among their preferred college choices. While selectivity varies by province, the sheer number of high school students applying for college each year has resulted in overall acceptance rates far lower than 0.1% of all test takers. Admission to Tsinghua's graduate schools is also very competitive. Only about 16% of MBA applicants are admitted each year.</p>
<p>Department of Mathematical Sciences
The Department of Mathematical Sciences (DMS) was established in 1927.
In 1952, Tsinghua DMS was merged with the Peking University Department of Mathematical Sciences. Then in 1979 it was renamed "Department of Applied Mathematics", and renamed again in 1999 to its current title.
Tsinghua DMS has three institutes at present, the institute of Pure Mathematics which has 27 faculty members, the Institute of Applied Mathematics and Probability and Statistics which has 27 faculty members, and the Institute of Computational Mathematics and Operations Research which has 20 faculty members. There are currently about 400 undergraduate students and 200 graduate students.</p>
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<h3>The Als3 protein can effectively adhere yeast to human intestinal epithelium</h3>
<p>We introduced the Als3 gene, regulated by a galactose promoter, into yeast and found that after the addition of galactose, it could attach in greater numbers to human intestinal epithelium compared to wild-type yeast.</p>
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<h2>Topic4</h2>
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<h3>Thiosulfate receptors can work effectively</h3>
<p>We introduced plasmids expressing downstream regulated GFP and thiosulfate receptors into yeast simultaneously and found that thiosulfate can significantly enhance the intensity and proportion of GFP signal expression in yeast.</p>
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<h3>Thiosulfate receptors can work effectively</h3>
<p>……to be continue</p>
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<h2id="Inhalation of muscone">
<h2>Inhalation of muscone</h2>
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<p>Tsinghua University is consistently ranked among the top universities in the Asia-Pacific according to major international university rankings. Tsinghua University ranked No. 1 in China, the whole of Asia-Oceania region and emerging countries according to the Times Higher Education, with its industry income, research, and teaching performance indicator placed at 1st, 4th and 9th respectively in the world. Internationally, Tsinghua was regarded as the most reputable Chinese university by the Times Higher Education World Reputation Rankings where, it has ranked 8th globally and 1st in the Asia-Pacific.</p>
<p>The Engineering Research Center for Navigation Technology is a relatively young institute in the Department of Precision Instrument which was established in 2000, with the intention to "[pursue] excellence in the research and development in the field of high-accuracy inertial instruments and navigation technology, as well as in MEMS inertial sensor fields, and to provide advanced training for future scientists and engineers in the field of inertial technology." Its research interests cover high-accuracy inertial instruments and navigation technology, MEMS inertial sensors and systems, and precise electro-mechanical control systems and their application. As of 2012, the area of the center is 2900 square meters, including approximately 550 square meters of clean rooms. Equipment and instruments in this center are worth over 50 million RMB (US$7.56 million).</p>
<p>We selected muscone gas molecules as the upstream control signal for our therapy system. We used the muscone receptor sequence from mouse olfactory epithelial cells, as employed by the Ye Haifeng team; for details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank">description</a>, and introduced it into the plasmid system expressed in Saccharomyces cerevisiae. We chose the mating pathway in Saccharomyces cerevisiae as the transmission pathway for the muscone signal within Saccharomyces cerevisiae. Based on the Benjamin M Scott team’s optimization, we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway, allowing the muscone receptor to be integrated into the Saccharomyces cerevisiae mating pathway.</p>
<p>We used the galactose promoter to induce the expression of the muscone signal receptor and the optimized Gα protein, and screened the successfully transformed Saccharomyces cerevisiae with a His nutritional deficiency. By controlling the induction conditions of galactose and muscone, we tested the effectiveness of the muscone gas molecule switch. For details, please refer to the protocol.</p>
<p>Aim：</p>
<p>Aim:</p>
<p>To validate the effectiveness of the muscone gas molecule switch in Saccharomyces cerevisiae.</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>
<p>Aim：</p>
<p>Aim:</p>
<p>To check the reporter signals downstream of the muscone molecular switch.</p>
<p>To check the synthesis of the secretion system downstream of the muscone molecular switch.</p>
<p>Lactic acid is the small molecule we selected to target the abnormally activated autoimmune cells in IBD diseases. For details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank">description</a>. We chose lactate dehydrogenase from E. coli to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize and secrete D-lactic acid. We used the galactose promoter to induce the expression of lactate dehydrogenase and screened the successfully transformed yeast with a Ura nutritional deficiency. By controlling the induction with galactose and glucose and establishing gradients of induction time and post-induction culture time, we tested the synthesis and secretion of lactic acid and searched for the optimal induction conditions for lactic acid secretion. For details, please refer to the protocol.</p>
<p>Aim：</p>
<p>Aim:</p>
<p>To test the effectiveness of the lactate secretion system in Saccharomyces cerevisiae.</p>
<p>To explore the optimal induction conditions for the lactate secretion system.</p>
<p>To avoid interference from the mating signals of Saccharomyces cerevisiae’s own growth on the signal transduction controlled by the muscone molecule and to ensure biological safety, we have made modifications to the genome of Saccharomyces cerevisiae. This is reflected in our knockout of the original receptor STE2 in the mating pathway of Saccharomyces cerevisiae. Our knockout system includes a gRNA targeting the STE2 gene and the Cas9 protein.</p>
<p>To avoid interference from the mating signals of Saccharomyces cerevisiae's own growth on the signal transduction controlled by the muscone molecule and to ensure biological safety, we have made modifications to the genome of Saccharomyces cerevisiae. This is reflected in our knockout of the original receptor STE2 in the mating pathway of Saccharomyces cerevisiae. Our knockout system includes a gRNA targeting the STE2 gene and the Cas9 protein.</p>
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<h3>Origin receptor knock-out</h3>
<p>We expressed the gRNA targeting the STE2 receptor gene and the Cas9 protein through a constitutive promoter and screened the successfully transformed yeast using leu nutritional deficiency. The genome of the successfully transformed Saccharomyces cerevisiae strains was sequenced to screen for strains with a successful knockout of STE2, followed by the transformation of the muscone molecular switch signaling pathway.</p>
<p>Aim：</p>
<p>To remove the interference of Saccharomyces cerevisiae’s own growth and mating signals on the secretory system.</p>
<p>Construct：STE2 gRNA&Cas9-pML107</p>
<p>Aim:</p>
<p>To remove the interference of Saccharomyces cerevisiae's own growth and mating signals on the secretory system.</p>
