Update TOC to support h3

.output/nitro.json

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The experiment was continued with the same ratio and then repeated with ratios of 1:3, 1:5, 1:6, and 1:9, but the construction was still unsuccessful."}]},{"type":"element","tag":"h3","props":{"id":"_828-914"},"children":[{"type":"text","value":"8.28-9.14"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"The construction of Pj23100-LuxI and Pj23100-Plux-LuxR-egfp was initiated, while still searching for the reasons behind the failure of recA-HRP-egfp construction. It was then decided to change the primers, adding protective bases at both ends of the primers' restriction sites."}]},{"type":"element","tag":"h3","props":{"id":"_915-929"},"children":[{"type":"text","value":"9.15-9.29"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Intein-mediated sequencing and ligation were performed, while continuously changing the primers, increasing the protective bases from one pair to three pairs, and repeating the construction of recA-HRP-egfp, but without success."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Ultimately, due to time constraints, the construction of recA-HRP-egfp was not successful, which prevented validation from being completed."}]}],"toc":{"title":"","searchDepth":1,"depth":3,"links":[{"id":"may-june","depth":2,"text":"May-June"},{"id":"june-september","depth":2,"text":"June-September","children":[{"id":"_623-626","depth":3,"text":"6.23-6.26"},{"id":"_626-630","depth":3,"text":"6.26-6.30"},{"id":"_72-75","depth":3,"text":"7.2-7.5"},{"id":"_76-710","depth":3,"text":"7.6-7.10"},{"id":"_710-713","depth":3,"text":"7.10-7.13"},{"id":"_713-724","depth":3,"text":"7.13-7.24"},{"id":"_724-827","depth":3,"text":"7.24-8.27"},{"id":"_828-914","depth":3,"text":"8.28-9.14"},{"id":"_915-929","depth":3,"text":"9.15-9.29"}]}]}},"_type":"markdown","_id":"content:wetlab-notebook.md","_source":"content","_file":"wetlab-notebook.md","_stem":"wetlab-notebook","_extension":"md"}
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+{"_path":"/project-description","_dir":"","_draft":false,"_partial":false,"_locale":"","title":"Description","description":"","body":{"type":"root","children":[{"type":"element","tag":"h2","props":{"id":"background"},"children":[{"type":"text","value":"Background"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Environmental endocrine disruptors (EDCs) are a class of chemical substances that can mimic or block the effects of hormones, affecting the normal development of organisms. These substances include DDT, dioxins, and phthalates, among others."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"..."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"DDT is an insecticide that was once widely used, but due to its harmful effects on the reproductive and nervous systems of organisms, as well as its potential carcinogenicity, its use has been restricted in many countries since the 1970s and 1980s. However, as these substances have already entered the environment in large quantities, their negative effects persist even though their production has stopped for many years. They accumulate in the atmosphere, soil, water bodies, and organisms, posing a natural hazard."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"In 2010, the direct social and economic loss caused by exposure to phthalate chemicals in China reached approximately 57.2 billion yuan, with the burden of male infertility being the most severe. Further calculations indicate that the total medical cost for all known EDCs exposure in China is about 429.4 billion yuan, accounting for approximately 1.07% of the annual GDP."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"..."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"All these indicate that we must pay attention to these potential dangers and have a more comprehensive understanding and early screening of environmental endocrine disruptors. Nowadays, with the deepening research on environmental endocrine disruptors, more and more studies are beginning to focus on the relationship between endocrine disruptors and their metabolites and other toxic effects. This superposition of multiple effects provides clues to revealing the molecular mechanisms underlying many diseases."}]},{"type":"element","tag":"h2","props":{"id":"problem"},"children":[{"type":"text","value":"Problem"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"In fact, the role of environmental endocrine disruptors in organisms is complex, and the inference of their adverse effects needs to emphasize the adverse outcome pathway (AOP pathway, which describes the logical derivation of causally related events at different levels of biological tissues, leading to adverse effects on human or wildlife health). This requires us to describe the molecular mechanisms behind the impact comprehensively."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Currently, the methods for detecting endocrine disruptors using biological effects mainly include competitive ligand binding assays, fused cell aggregation experiments, and animal cell proliferation analysis experiments. These methods can only evaluate the endocrine disruption effect through the binding effect of hormone receptors when analyzing endocrine disruptors, and cannot judge the involvement of other effects behind the biological damage caused by endocrine disruptors, therefore, they have limitations."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"More and more literature has proven the important role of oxidative damage effects in the damage caused by environmental endocrine disruptors to organisms. For example, exposure to various parabens (PBs) and their metabolites can increase the level of oxidative stress, and the superposition of oxidative damage and endocrine disruption increases the risk of impaired reproductive capacity in organisms. The collaborative analysis of environmental endocrine disruption effects and oxidative damage effects can help us predict the pathways of action of environmental endocrine disruptors on organism damage, thereby assisting us in having a clearer understanding of their damage consequences and providing a reference for regulatory authorities to implement targeted regulatory policies."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Our project is based on this concept and aims to construct microbial sensors through synthetic biology methods to provide multiple detection lines for the assessment of endocrine disruption effects and oxidative damage effects. Not only can we achieve the separate detection of the two effects, but we can also realize the collaborative analysis of the two effects through logic gate detection lines, achieving a rapid, convenient, and comprehensive assessment of environmental endocrine disruptors. Conduct more in-depth research and discussion on the impact mechanism of environmental endocrine disruptors."}]},{"type":"element","tag":"h2","props":{"id":"inspiration"},"children":[{"type":"text","value":"Inspiration"}]},{"type":"element","tag":"h3","props":{"id":"biology"},"children":[{"type":"text","value":"biology"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Initially, we planned to develop a biosensor for detecting environmental endocrine disruptors. To achieve this goal, we studied the characteristics and mechanisms of action of environmental endocrine disruptors and researched the methods for detecting them. Intein peptides are protein analogs found in self-splicing RNA introns, and their insertion can lead to the inactivation of the host protein. Typically, they excise themselves from the host cell after translation, restoring the activity of the host protein. By replacing the homing endonuclease domain in the intein peptide with a hormone receptor binding domain, we can construct a hormone-sensitive intein peptide, which can be used to create recognition elements for endocrine disruption effects. We plan to construct an estrogen-sensitive intein peptide and insert it into T7 RNA Polymerase. T7 RNA Polymerase can specifically activate the T7 promoter, thus regulating the downstream circuit to express the reporter gene upon detection of estrogen mimics through the T7 promoter."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Subsequently, through more in-depth researches, it was found that the effects of environmental endocrine disruptors in organisms are complex. An increasing number of studies have demonstrated the significant role of oxidative stress in the damage caused by environmental endocrine disruptors. For example, exposure to various paraben compounds (PBs) and their metabolites can increase oxidative stress levels. The combined oxidative damage and endocrine disruption can heighten the risk of impaired reproductive capability in organisms. A synergistic analysis of the effects of environmental endocrine disruption and oxidative damage can help us predict the pathways through which these disruptors cause harm to organisms, thereby assisting us in gaining a clearer understanding of their damaging consequences. Therefore, we plan to incorporate DNA damage detection, designing an and gate detection circuit capable of simultaneously detecting endocrine disruption effects and DNA oxidative damage effects."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"DNA damage detection is based on the SOS response mechanism in "},{"type":"element","tag":"em","props":{},"children":[{"type":"text","value":"Escherichia coli"}]},{"type":"text","value":", which is an inducible DNA repair system activated when bacterial DNA is damaged. When bacteria detect DNA damage, the expression levels of genes associated with the SOS response mechanism significantly increase, leading to the expression of proteases that relieve the repression of target proteins, initiating the repair process. We chose the recA promoter to detect DNA damage, when bacteria sense DNA damage, the expression level of this promoter rises significantly, thereby activating downstream reporter genes."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"To construct the logic gate, we introduced the hrp system, which consists of the hrpR and hrpS genes along with the HrpL promoter regulated by the sigma54 factor. The hrpL promoter is activated only when both hrpR and hrpS genes are expressed simultaneously."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"We plan to insert the hrpR gene downstream of the P recA promoter, which will be activated in response to DNA damage effects; the hrpS gene will be inserted downstream of the P T7 promoter, which will be activated after the peptide-specific binding domain binds to estrogen analogs. The reporter gene egfp will be inserted downstream of the P hrpL promoter, resulting in a two-input AND gate circuit."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"However, the entire circuit is too large, posing significant challenges to the stability of the plasmid and the burden on the engineered bacteria. Therefore, we plan to divide the entire circuit into two parts, each constructed in different engineered bacteria, inserting new components to enable signal transmission between the two engineered bacterias."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Throughout the system, we have always planned to use the egfp gene as the reporter gene. However, this gene requires the presence of oxygen molecules as a cofactor for its luminescence, while the cultivation chamber of our designed hardware is in a low-oxygen environment. As a result, we tested a new reporter gene, BsFbFp, which can express significantly enhanced in vivo fluorescence in both aerobic and anaerobic environments, ensuring that the engineered bacteria can effectively express fluorescent signals in low-oxygen conditions."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Thus, the construction of the biological part of our circuit has been completed."}]},{"type":"element","tag":"h3","props":{"id":"hardware"},"children":[{"type":"text","value":"Hardware"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Point of Care Testing (POCT) is a form of rapid on-site testing/near-patient testing method which has been particularly impactful and is playing an increasingly vital role in the fields of medical diagnostics and environmental health monitoring due to its speed, convenience, and ease of operation. Traditional water contaminant detection technologies are often constrained by the need for costly equipment and a professional laboratory environment. In contrast, novel biosensor detection devices based on compact POCT equipment, when connected to an interactive app for operational control and visual data analysis, can effectively facilitate the rapid and efficient screening of emerging waterborne pollutants. This approach holds broad prospects for application in the field of environmental monitoring and assessment."}]},{"type":"element","tag":"h2","props":{"id":"solution"},"children":[{"type":"text","value":"Solution"}]},{"type":"element","tag":"h3","props":{"id":"biology-1"},"children":[{"type":"text","value":"biology"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"To achieve signal communication between the two engineered bacteria, we studied the bacterial quorum sensing mechanism. This mechanism allows bacteria to exchange information and coordinate population behaviors when their numbers reach a certain density, regulating gene expression. When activated, bacteria secrete signal molecules that can freely diffuse across the cell membrane. Once the concentration of these signal molecules in the environment reaches a certain threshold, it induces the expression of specific genes in the bacteria. We introduced the LuxI/R system, where AHL (N-acyl homoserine lactone) is produced catalytically by the expression of the LuxI gene as a signaling molecule. This AHL freely diffuses into the second engineered bacteria and binds with the LuxR protein released by the activation of the LuxR gene, activating the P LuxR promoter to initiate downstream gene expression."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/description/biocircuit-p1.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/description/biocircuit-p2.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/description/biocircuit-p3.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/description/biocircuit-p4.png"},"children":[]}]},{"type":"element","tag":"h3","props":{"id":"hardware-1"},"children":[{"type":"text","value":"Hardware"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"In response to the demands for standardized cultivation of engineered bacteria and high-sensitivity detection, we have designed a highly integrated and automated system that encompasses the entire process of bacterial strain revival, cultivation, detection, and analysis. The system utilizes an ESP32 chip as the main controller, interfaces with a temperature control system and a fluorescence detection system, employs a linear slide rail structure for high-throughput detection, and leverages a mobile app for automated device control and visualization of detection results. This system overcomes the limitations of time and space associated with traditional detection equipment, providing a versatile, portable, and highly sensitive solution for the detection of EDCs in aquatic environments across various scenarios."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/description/and-gate.png"},"children":[]}]},{"type":"element","tag":"h2","props":{"id":"reference"},"children":[{"type":"element","tag":"em","props":{},"children":[{"type":"text","value":"Reference"}]}]},{"type":"element","tag":"mdc-wrap","props":{":class":"reference"},"children":[{"type":"element","tag":"ol","props":{},"children":[{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Cao Y, Li L, Shen KH, Liu JG. Disease burden attributable to endocrine-disrupting chemicals exposure in China: A case study of phthalates. Science of the Total Environment, 2019, Vol. 662(1): 615-621"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Nematollah Asadi, Mahmoud Bahmani, Arash Kheradmand, Mahmoud Rafieian-Kopaei. The Impact of Oxidative Stress on Testicular Function and the Role of Antioxidants in Improving it: A Review. Journal of Clinical and Diagnostic Research, 2017, Vol. 11(5): IE1-IE5"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Ma Ya, Liu Haohao, Wu Jinxia, Yuan Le, Wang Yueqin, Du Xingde, Wang Rui, Marwa Phelisters Wegesa, Petlulu Pavankumar, Chen Xinghai, Zhang Huizhen. The adverse health effects of bisphenol A and related toxicity mechanisms. Environmental Research, 2019, Vol. 176: 108"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Liang R , Zhou J , Liu J. Construction of a Bacterial Assay for Estrogen Detection Based on an Estrogen-Sensitive Intein. Applied & Environmental Microbiology, 2011, 77(7):2488.DOI:10.1128/AEM.02336-10."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Ma Ya, Liu Haohao, Wu Jinxia, Yuan Le, Wang Yueqin, Du Xingde, Wang Rui, Marwa, Phelisters Wegesa, Petlulu, Pavankumar, Chen Xinghai, Zhang Huizhen.The adverse health effects of bisphenol A and related toxicity mechanisms.Environmental Research, 2019, Vol.176: 108575"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"LIANG Jing-jia, GU Ai-hua. Multigenerational and cross-generational effect of environmental endocrine disruptors on reproductive system in male animals. "},{"type":"element","tag":"em","props":{},"children":[{"type":"text","value":"Chinese Journal of Public Health"}]},{"type":"text","value":", 2021, 37(2): 375-380."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Nematollah Asadi, Mahmoud Bahmani, Arash Kheradmand, Mahmoud Rafieian-Kopaei.The Impact of Oxidative Stress on Testicular Function and the Role of Antioxidants in Improving it: A Review.Journal of Clinical and Diagnostic Research, 2017, Vol.11(5): IE1-IE5"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Mondal Shirsha, Ghosh Songita, Bhattacharya Samir, Mukherjee Sutapa. Chronic dietary administration of lower levels of diethyl phthalate induces murine testicular germ cell inflammation and sperm pathologies: Involvement of oxidative stress. Chemosphere, 2019, Vol.229: 443-451"}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Maslowska K H, Karolina Makiela‐Dzbenska, Fijalkowska I J. The SOS system: A complex and tightly regulated response to DNA damage. John Wiley & Sons, Ltd, 2019(4). DOI:10.1002/em.22267."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Baojun W, Mauricio B, Martin B. Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks. Nucleic Acids Research, 2014(14): 9484-9492. DOI:10.1093/nar/gku593."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Xiao Y, Zou H, Li J, Song T, Lv W, Wang W, Wang Z, Tao S. Impact of quorum sensing signaling molecules in gram-negative bacteria on host cells: current understanding and future perspectives. Gut Microbes. 2022 Jan-Dec, 14(1):2039048. doi: 10.1080/19490976.2022.2039048."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Drepper T, Eggert T, Circolone F, et al. Reporter proteins for in vivo fluorescence without oxygen. Nature Biotechnology, 2007, 25(4):443-445.DOI:10.1038/nbt1293."}]},{"type":"element","tag":"li","props":{},"children":[{"type":"text","value":"Mukherjee A, Schroeder C M, Sikes H D, et al. Flavin-based fluorescent proteins: emerging paradigms in biological imaging. "},{"type":"element","tag":"em","props":{},"children":[{"type":"text","value":"This review comes from a themed issue on Analytical biotechnology"}]},{"type":"text","value":". 2015."}]}]}]}],"toc":{"title":"","searchDepth":1,"depth":3,"links":[{"id":"background","depth":2,"text":"Background"},{"id":"problem","depth":2,"text":"Problem"},{"id":"inspiration","depth":2,"text":"Inspiration","children":[{"id":"biology","depth":3,"text":"biology"},{"id":"hardware","depth":3,"text":"Hardware"}]},{"id":"solution","depth":2,"text":"Solution","children":[{"id":"biology-1","depth":3,"text":"biology"},{"id":"hardware-1","depth":3,"text":"Hardware"}]},{"id":"reference","depth":2,"text":"Reference"}]}},"_type":"markdown","_id":"content:project-description.md","_source":"content","_file":"project-description.md","_stem":"project-description","_extension":"md"}
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+{"_path":"/wetlab-results","_dir":"","_draft":false,"_partial":false,"_locale":"","title":"Results","description":"Endocrine disruptors are a class of chemicals that can interfere with the normal function of the endocrine system, and they may mimic or inhibit the effects of natural hormones, thereby affecting the physiological processes of the body. These substances are widely found in plastics, pesticides, industrial chemicals, and daily consumer goods. They enter the human body through the food chain or through direct contact, interfering with hormonal balance, and may cause reproductive health problems, developmental disorders, metabolic disorders and even some types of cancer.","body":{"type":"root","children":[{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Endocrine disruptors are a class of chemicals that can interfere with the normal function of the endocrine system, and they may mimic or inhibit the effects of natural hormones, thereby affecting the physiological processes of the body. These substances are widely found in plastics, pesticides, industrial chemicals, and daily consumer goods. They enter the human body through the food chain or through direct contact, interfering with hormonal balance, and may cause reproductive health problems, developmental disorders, metabolic disorders and even some types of cancer."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"When endocrine disruptors affect hormone activity, they may also induce cellular oxidative stress because they increase the generation of reactive oxygen species (ROS), highly reactive chemicals that damage cellular structures, including proteins, lipids, and DNA. Long-term oxidative stress accelerates cellular aging and may also lead to male infertility and the development of multiple chronic diseases. Thus, there is a close link between exposure to endocrine disruptors and cellular oxidative damage, a potential promoter to multiple health problems. Therefore, it is necessary to detect the endocrine interference effects of endocrine disruptors and oxidative damage effects in the relevant location samples."}]},{"type":"element","tag":"h2","props":{"id":"_1-puc19-preca-bsfbfp-bl21"},"children":[{"type":"text","value":"1. pUC19-PrecA-BsFbFp-BL21"}]},{"type":"element","tag":"h3","props":{"id":"strain-construction"},"children":[{"type":"text","value":"Strain construction"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"We plan to design a portable microbial sensor, in which the sensor chamber will be maintained in a nearly anoxic state. Under anoxic conditions, the fluorescence intensity of EGFP diminishes, and its detection limit decreases; therefore, traditional EGFP is not the optimal choice for our application. We have constructed the PrecA-BsFbFP circuit, where BsFbFP is an anoxic fluorescent protein that exhibits fluorescence intensity nearly equivalent to that of EGFP under anoxic conditions, while significantly improving the detection limit. Consequently, we will utilize BsFbFP as the expression product of our engineered strain for detection characterization."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"We obtained the PrecA-BsFbFp circuit and the recA-eGFP (hereafter referred to as wild) circuit through PCR.The gene circuits were ligated to the pUC19 vector using enzyme ligation and transformed into BL21 competent cells . The transformed cells were spread onto LB solid medium supplemented with ampicillin. Single colonies that grew were selected for amplification and sent to Jinweizhi (Suzhou) Biochemistry Co. for sequencing, and the remaining bacteria were preserved in glycerol."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/fig1.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/2-enzyme-digestion.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 1. Results of the enzyme digestion verification experiment for pUC19-PrecA-BsFbFP-BL21."}]}]},{"type":"element","tag":"h3","props":{"id":"characterization-of-experiments"},"children":[{"type":"text","value":"Characterization of experiments"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Firstly, we performed screening of the pUC19-PrecA-BsFbFp-BL21. The transformed wild-type bacteria were cultured on a shaker until the OD value reached 0.2, followed by the addition of NA for induction for 2 h. In aerobic conditions, BsFbFp is significantly less characterized than EGFP, while the RFU is close, and BsFbFp has a high detection limit, indicating that BsFbFp is an excellent choice for characterization in anaerobic conditions."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/o2-fluo.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/o2-rfu.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 2. Characterization results of BsFbFP and EGFP under anoxic and NA gradient injury conditions."}]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"We have characterized the pUC19-PrecA-BsFbFp-BL21 under anaerobic conditions.The transformed wild-type bacteria were cultured on a shaker until the OD value reached 0.2, followed by the addition of NA for induction for 2 h.As the concentration of NA increased, the fluorescence intensity of engineering strains increased accordingly. Several experiments proved that the RFU value increased with the concentration of NA in the anaerobic environment."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/10-rfu.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/10-rfu-more.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 3. Fluorescence intensity characterization of BsFbFP under NA gradient injury conditions in 10-lianpao chip cultures, expressed as RFU."}]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"Later, we compared the characterization difference between DH5α strain and BL21 strain, and showed that the BL21 strain has higher sensitivity to injury, reflecting its advantage as a representative strain."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/o2-fluo-more.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 4. Fluorescence characterization gradients of DH5α and BL21 under aerobic and NA gradient injury conditions."}]}]},{"type":"element","tag":"h2","props":{"id":"_2-pet28-preca-hrpr-hrps-phrpl-bsfbfp-bl21"},"children":[{"type":"text","value":"2. pET28-PrecA-hrpR-hrpS-PhrpL-BsFbFp-BL21"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"The gene circuits were ligated to the pET28 vector using enzyme ligation and transformed into BL21 competent cells . The transformed cells were spread onto LB solid medium supplemented with ampicillin.Preserved strains were stored in glycerol."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/hrp.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/2-enzyme-digestion.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 5. Results of the enzyme digestion verification experiment for pET28-PrecA-hrpR-hrpS-PhrpL-BsFbFp-BL21."}]}]},{"type":"element","tag":"h2","props":{"id":"_3-puc19-pbad-n-t7rnap-er-lbd-intein-c-t7rnap-bsfbfp"},"children":[{"type":"text","value":"3. pUC19-PBAD-N-T7RNAP-ER-LBD intein-C-T7RNAP-BsFbFp"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"The gene sequencing conducted by our company has successfully validated the synthesis of the intein of the biological circuit."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/fig3.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/gene-squencing.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 6. Gene sequencing results of the ER-LBD intein."}]}]},{"type":"element","tag":"h2","props":{"id":"_4-psb1c3-pj23100-luxi-bl21-and-psb1c3-plux-luxr-egfp-bl21"},"children":[{"type":"text","value":"4. psB1C3-Pj23100-LuxI-BL21 and psB1C3-Plux-LuxR-EGFP-BL21"}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/fig4.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"text","value":"We obtained by PCR the Pj233100, Plux-LuxR, LuxI, and EGFP and ligated it to the psB1C3 plasmid into E. coli BL21. The transformed cells were spread onto LB solid medium supplemented with chloramphenicol. Recombinants were used for characterization experiments and stored in glycerol."}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/pcr.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 7. PCR products of the Pj23100-LuxI fragment and the Plux-LuxR-EGFP fragment."}]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/lux-digestion.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 8. Enzyme digestion verification of psB1C3-Pj23100-LuxI-BL21 and Plux-LuxR-EGFP-BL21."}]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"img","props":{"alt":"img","src":"https://static.igem.wiki/teams/5358/experiment/ligation.png"},"children":[]}]},{"type":"element","tag":"p","props":{},"children":[{"type":"element","tag":"span","props":{"className":["fig-title"]},"children":[{"type":"text","value":"Figure 9. Enzyme Ligation Product of psB1C3-Pj23100-LuxI-BL21 and Plux-LuxR-EGFP-BL21."}]}]}],"toc":{"title":"","searchDepth":1,"depth":3,"links":[{"id":"_1-puc19-preca-bsfbfp-bl21","depth":2,"text":"1. pUC19-PrecA-BsFbFp-BL21","children":[{"id":"strain-construction","depth":3,"text":"Strain construction"},{"id":"characterization-of-experiments","depth":3,"text":"Characterization of experiments"}]},{"id":"_2-pet28-preca-hrpr-hrps-phrpl-bsfbfp-bl21","depth":2,"text":"2. pET28-PrecA-hrpR-hrpS-PhrpL-BsFbFp-BL21"},{"id":"_3-puc19-pbad-n-t7rnap-er-lbd-intein-c-t7rnap-bsfbfp","depth":2,"text":"3. pUC19-PBAD-N-T7RNAP-ER-LBD intein-C-T7RNAP-BsFbFp"},{"id":"_4-psb1c3-pj23100-luxi-bl21-and-psb1c3-plux-luxr-egfp-bl21","depth":2,"text":"4. psB1C3-Pj23100-LuxI-BL21 and psB1C3-Plux-LuxR-EGFP-BL21"}]}},"_type":"markdown","_id":"content:wetlab-results.md","_source":"content","_file":"wetlab-results.md","_stem":"wetlab-results","_extension":"md"}
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