1.Through literature review and with expert assistance, two dynamic regulation systems were identified: Thermosensitive switch system & Protein encapsulation system.

2.Commissioned a company to synthesize the J23100-RBS-TlpA39-PtlpA1 sequence, and the 0.5A-IDP and 1.0A-IDP gene fragments.

1.Discussed the selection of mutation sites with dry lab team members.

2.Designed primers required for mutagenesis.

1.Screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) enzymes from different organisms: from Corynebacterium glutamicum ATCC 13032 (CggapC), Bacillus subtilis (BsrocG), Clostridium saccharobutylicum DSM 13864 (CsgapC), and Streptococcus mutans (SmgapN).

2.Obtained the BsrocG gene fragment via genomic extraction; the other three were synthesized by a company.

1.Constructed genetic circuits for the Thermosensitive switch system: PJ23100-RBS-TlpA39-PtlpA1-RBS-mWasabi and PJ23100-RBS-TlpA39-PtlpA1-RBS-CadA, using the vector pET28a(ΔlacI).

2.Constructed genetic circuits for the Protein encapsulation system: mWasabi-TEV-site-0.5A-IDP, mWasabi-TEV-site-1.0A-IDP, CadA-TEV-site-0.5A-IDP, CadA-TEV-site-1.0A-IDP, CadA-TVMV-site-0.5A-IDP, CadA-TVMV-site-1.0A-IDP, using the vector pCDFDuet under the Ptrc promoter.

3.Transformed the recombinant plasmids into E. coli DH5α competent cells. Selected single colonies for colony PCR verification. Colonies with correct amplification band sizes were chosen, their plasmids were extracted and sent for sequencing verification.

1.Constructed combinatorial mutant plasmids. Used overlap extension PCR (overlap-PCR) to amplify gene fragments covering all mutation sites. These fragments were connected to the plasmid backbone DNA fragment via homologous recombination to obtain the mutant plasmids: pCDFDuet-Ptrc-CadA^{H245Q,W333E,E526N,K527S,G655F}, pCDFDuet-Ptrc-CadA^{H245Q,W333H,E526N,K527S,G655F}, pCDFDuet-Ptrc-CadA^{H245Q,W333H,E526N,K527S,G655K}, pCDFDuet-Ptrc-CadA^{H245Q,W333E,E526N,K527S,G655K}, pCDFDuet-Ptrc-CadA^{H245F,W333E,E526N,K527S,G655F}.

2.Transformed these into E. coli DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

1.Cloned the four GAP gene fragments into the linearized pCDFDuet-Ptrc vector using In-fusion cloning, resulting in plasmids pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, pCDFDuet-Ptrc-SmgapN.

2.Transformed these into E. coli DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

1.All plasmids were successfully constructed.

2.Transformed the sequence-verified plasmids into E. coli NT1003.

3.Picked colonies from the plate, inoculated them into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

4.After starting fermentation, periodically measured fluorescence intensity, glucose, and L-lysine levels. When glucose was depleted, switched to anaerobic fermentation.

1.Transformed the sequence-verified plasmids into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

4.All combinatorial mutants lost enzyme activity. Decided to proceed with single-point mutations based on analysis.

1.Transformed the sequence-verified plasmids into E. coli NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

1.Analyzed and organized experimental data. Found that the Protein encapsulation system failed to effectively encapsulate the CadA enzyme, allowing it to leak and catalyze the conversion of L-lysine, preventing L-lysine accumulation. However, the fluorescence intensity of the 1.0 A-IDP group was significantly lower than that of the 0.5 A-IDP group, indicating that 1.0 A-IDP has better encapsulation efficiency for the fluorescent protein compared to 0.5 A-IDP.

2.Based on expert advice to enlarge the A-IDP size, attempted to commission synthesis from a company, but failed due to excessive repetitive sequences. Decided to construct it ourselves.

1.Constructed alanine mutant plasmids for eight sites: I182, H245, K246, W333, E526, K527, Y652, G655. We designed point mutation primers and cloned the whole plasmid using PCR. The linearized plasmid was isolated and purified by agarose gel electrophoresis.

2.Transformed these into E. coli DH5α competent cells, extracted plasmids, and sent them for sequencing verification.

1.Measured the activity of the NADP⁺-dependent GAP enzymes by detecting the succinate produced from last week's fermentation samples using HPLC.

2.Analyzed and organized experimental data. All four dehydrogenases showed dehydrogenase activity. Among them, Cggap from Clostridium saccharobutylicum showed the highest dehydrogenase efficiency. However, the succinate concentration was much lower than expected.

1.Attempted to self-construct the 2.0A-IDP plasmid: Using 1.0A-IDP as a template to synthesize the larger 2.0 A-IDP. Digested the pCDFDuet-Ptrc-cadA-TEV/TVMV-site-1.0A-IDP vector plasmid with KpnI and SalI. Then PCR-amplified the 1.0AIDP fragment with primers containing KpnI and SalI sites. Ligated using T4 DNA Ligase.

2.Transformed the recombinant product into E. coli DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

The previously failed G655A construct was re-attempted, but the result was still unsuccessful.

1.Decided to switch to the low-copy-number plasmid pACYCDuet.

2.Using plasmids pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, pCDFDuet-Ptrc-SmgapN as templates, PCR-amplified the CggapC, BsrocG, CsgapC, and SmgapN fragments. Used In-fusion cloning to perform homologous recombination of CggapC, BsrocG, CsgapC, and SmgapN with the linearized pACYCDuet vector, respectively.

3.Transformed the recombinant products into E. coli DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

1.Transformed the plasmid pCDFDuet-Ptrc-CadA-TEV/TVMV-site-2.0A-IDP into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

4.Results showed no L-lysine accumulation, suggesting construction failure. Decided to abandon the Protein encapsulation system strategy. Also observed that cadaverine accumulation in the Thermosensitive switch system gradually decreased as temperature increased, decided to optimize and improve this later.

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadA^I182A, pCDFDuet-Ptrc-CadA^H245A, pCDFDuet-Ptrc-CadA^K246A, pCDFDuet-Ptrc-CadA^W333A, pCDFDuet-Ptrc-CadA^E526A, pCDFDuet-Ptrc-CadA^K527A, pCDFDuet-Ptrc-CadA^Y652A, and the unmutated original plasmid into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

1.Transformed the sequence-verified plasmids into E. coli NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion

1.Received expert advice to control the regulation of TlpA39 from the promoter aspect. Selected promoters BBa_J23106 and BBa_J23119 from the part library to replace and compare with the original promoter BBa_J23100.

2.Replaced the promoter PJ23100 in pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-cadA with PJ23106 and PJ23119.

3.Transformed the recombinant products into E. coli DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

1.Selected I182, H245, K246, which did not completely lose activity, for subsequent mutation to E, L, S, F, V. Constructed the corresponding mutant plasmids.

2.Transformed the recombinant products into E. coli DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

3.Sequencing showed the K246V construct failed. Repeated construction was still unsuccessful.

1.Measured performance by detecting succinate produced by recombinant E. coli using HPLC.

2.Results showed that after recombinant expression in the low-copy-number plasmid pACYCDuet, CggapC from Clostridium saccharobutylicum again showed increased succinate production, while L-lysine yield remained stable. Therefore, the CggapC gene was selected for constructing the fermentation strain going forward.

1.Transformed plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-cadA, pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-cadA, pET28a(ΔI7)-PJ23119-RBS-TlpA39-PtlpA-cadA into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadA^I182E, pCDFDuet-Ptrc-CadA^I182L, pCDFDuet-Ptrc-CadA^I182S, pCDFDuet-Ptrc-CadA^I182F, pCDFDuet-Ptrc-CadA^I182V, pCDFDuet-Ptrc-CadA^H245E, pCDFDuet-Ptrc-CadA^H245L, pCDFDuet-Ptrc-CadA^H245S, pCDFDuet-Ptrc-CadA^H245F, pCDFDuet-Ptrc-CadA^H245V, pCDFDuet-Ptrc-CadA^K246E, pCDFDuet-Ptrc-CadA^K246L, pCDFDuet-Ptrc-CadA^K246S, pCDFDuet-Ptrc-CadA^K246F, and the unmutated original plasmid into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C

3.Collected cells by centrifugation and performed whole-cell catalysis.

1.Following Dr. Xiao-Jie Guo's suggestion, we chose to overexpress the soluble pyridine nucleotide transhydrogenase (sthA) from E. coli MG1655 to promote rational allocation and cycling of cofactors.

2.Subcloned the sthA gene fragment together with CggapC into the linearized pACYCDuet vector, generating the pACYCDuet-sthA-CggapC plasmid.

3.Transformed the recombinant product into E. coli DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

1.Analyzed and organized experimental data. Identified PJ23106 as the optimal promoter. The issue of decreasing cadaverine accumulation with increasing temperature was alleviated to some extent but still needed improvement.

2.Learned from experts that RNA thermometers can refine the temperature response gradient. After literature review, selected U7 and U8 for insertion into our plasmids.

3.Commissioned a company to synthesize the U7 and U8 gene sequences.

1.Analyzed and organized experimental data; no mutations resulted in increased enzyme activity.

2.Conducted literature review and in-depth discussions with experts and the dry lab team. Began attempting to re-predict potential beneficial mutations from the perspective of decamer stability.

1.Transformed the sequence-verified plasmids into E. coli NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

1.Constructed plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U8-cadA.

2.Transformed into E. coli DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

1.Obtained F14C/K44C, V12C/K41C, F14C/L45C, L93C/E445C, Y13C/P36C, E104K via computational simulation analysis, and included V12C/K44C, F14C/D41C, L89C/E445C from literature for mutational.

2.Transformed into E. coli DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

3.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadA^F14C/K44C, pCDFDuet-Ptrc-CadA^V12C/K41C, pCDFDuet-Ptrc-CadA^F14C/L45C, pCDFDuet-Ptrc-CadA^L93C/E445C, pCDFDuet-Ptrc-CadA^Y13C/P36C, pCDFDuet-Ptrc-CadA^E104K, pCDFDuet-Ptrc-CadA^V12C/K44C, pCDFDuet-Ptrc-CadA^F14C/D41C, pCDFDuet-Ptrc-CadA^L89C/E445C, and the unmutated original plasmid into E. coli BL21.

1.Measured performance by detecting L-lysine and succinate produced by E. coli using HPLC.

2.Results showed that the strain with pACYCDuet-sthA-CggapC increased succinate production by 26.3% compared to the empty vector, and by 70% compared to the pACYCDuet-CggapC strain, while L-lysine production remained stable. We decided to use this strain in subsequent experiments.

1.Transformed plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-U7-cadA, pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-cadA, pET28a(ΔI7)-PJ23119-RBS-TlpA39-PtlpA-cadA (control) into E. coli NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

1.Picked single colonies from the plate, inoculated into 5 mL LB seed medium, and cultured for 8h. Then transferred at 1% inoculation volume into 50 mL LB medium for expansion. When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce target protein expression, and continued induction culture overnight at 37°C.

2.Collected cells by centrifugation and performed whole-cell catalysis.

3.Identified Y13C/P36C as the optimal mutant. Performed iterative mutations, constructing mutants Y13C/P36C/E445Q, Y13C/P36C/K477R, Y13C/P36C/L93C/E445C.

4.Transformed into E. coli DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

1.Analyzed and organized experimental data. Found that leaky expression significantly decreased after adding U7/U8.

2.Replaced the unmutated CadA in the selected optimal mutant Y13C/P36C within the plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-cadA.

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadA^{Y13C/P36C/E445Q}, pCDFDuet-Ptrc-CadA^{Y13C/P36C/K477R}, pCDFDuet-Ptrc-CadA^{Y13C/P36C/L93C/E445C}, and the unmutated original plasmid into E. coli BL21.

2.Picked single colonies from the plate, inoculated into 5 mL LB seed medium, and cultured for 8h. Then transferred at 1% inoculation volume into 50 mL LB medium for expansion. When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce target protein expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

4.Analyzed and organized experimental data.

1.Based on previous experiments, we finalized the optimized Thermosensitive switch system and the Cofactor conversion system. Subsequently, we introduced the plasmids for these two modules into the L-lysine-producing E. coli NT1003 to construct a cell factory capable of co-producing cadaverine and succinate.

2.Co-transformed plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U8-cadA respectively with plasmid pACYCDuet-sthA-CggapC into the L-lysine producing strain E. coli to construct cell factories capable of co-producing cadaverine and succinate.

3.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 33°C for over 20 hours.

4.Periodically measured glucose and L-lysine levels. After glucose depletion, cultured at 42°C for 4 hours to activate the thermosensitive switch, then switched to anaerobic fermentation.

1.Measured performance by detecting cadaverine and succinate produced by E. coli using HPLC.

2.Fermentation results showed that the recombinant strains successfully achieved synergistic synthesis of cadaverine and succinate, with yields reaching 5.7 g/L and 5.4 g/L, respectively. Through precise metabolic flux regulation, the molar ratio of the two products approached the ideal 1:1. This created excellent thermodynamic conditions for the subsequent acid-base neutral reaction to generate PA54 salt, eliminating the need for additional acid or base, simplifying steps and reducing costs. Furthermore, from the perspective of carbon metabolic flux, the matched yields of the two products indicated a good balance between CO₂ fixation and release in their biosynthetic pathways, improving overall carbon economy and laying a solid foundation for the efficient and green preparation of the polymer monomer PA54.

Environment-responsive enzyme-controlled release system: Established an ODE model from signal sensing to product formation and compiled key parameters for CadC signal perception and cadBA transcriptional regulation.

Cadaverine-producing enzyme model: Screened key active sites of CadA and performed molecular docking, preliminarily identifying potential engineering targets.

Metabolic flux study: Reviewed project background and challenges to lay the foundation for subsequent modeling.

Environment-responsive system: Refined transcriptional regulation and protein release modules, integrated metabolic conversion kinetics, and ensured mass balance.

Cadaverine enzyme model: Designed the deep learning model architecture and constructed a data pipeline to extract features from protein sequences and compound graph structures.

Metabolic flux study: Studied CadA directed evolution literature to inform the inclusion of enzyme constraints and regulatory logic in models.

Environment-responsive system: Integrated the four modules into a unified ODE system; initial simulation showed two-stage dynamics: aerobic accumulation and anaerobic production.

Cadaverine enzyme model: Implemented feature extraction and custom DataLoader to prepare for training.

Metabolic flux study: Installed and tested metabolic modeling software, clarifying dry-lab tasks.

Environment-responsive system: Validated and calibrated the model, designed parameter sensitivity analysis, and developed visualization tools.

Cadaverine enzyme model: Implemented feature fusion and activity prediction modules with early stopping to prevent overfitting. Lysine decarboxylase docking simulation to identify key amino acid residues.

Metabolic flux study: Set up the modeling environment, including Anaconda, Gurobi, and essential libraries.

Environment-responsive system: Conducted sensitivity analysis, quantified performance metrics, and predicted cadaverine yield under optimal conditions.

Cadaverine enzyme model: Prepared kcat dataset and configured the training environment.

Metabolic flux study: Learned FBA and FVA, and tested the iML1515 model.

Environment-responsive system: Simulated the effects of different A-IDP encapsulation sizes.

Cadaverine enzyme model: Trained the generalized model and performed preliminary performance evaluation.

Metabolic flux study: Built a project-specific aerobic model including cadaverine and related reactions.

Metabolic flux study: Constructed anaerobic model, added succinate-related reactions, and performed baseline flux analysis.

Cadaverine enzyme model: Fine-tuned the model using a specific dataset to improve prediction accuracy.

Metabolic flux study: Performed FVA and FSEOF analyses to identify key target gene gapA.

Cadaverine enzyme model: Designed genetic algorithms to screen high-activity mutants and discussed candidate results with the wet-lab team.

Metabolic flux study: Compiled analysis reports, submitted prioritized targets, and compared predictions with wet-lab validation data.

Cadaverine enzyme model: Organized code, model weights, and predictions; completed iGEM Wiki documentation; summarized methodological innovations and compared computational predictions with wet-lab enzyme activity.

1. Conducted team brainstorming sessions to define our project focus on bio-based nylon development.

2. Interviewed expert Cao Min from Kingfa Science & Technology to understand the market landscape for bio-based polyamides and identify the technological gap for fully bio-based short-chain polyamides.

3. Consulted Professor Chen Kequan to learn about bio-based material synthesis technologies available in the university lab, leading to the final decision to pursue the "Single-cell Synchronous Synthesis of PA54" project.

4. Held discussions with representatives from China National Petroleum Corporation (CNPC) to clarify the environmental, economic, and resource-related challenges within the conventional fossil-based nylon industry.

5. Initiated public awareness research on bio-based materials by designing online and offline survey distribution plans.

1. Prepared training materials on iGEM competition rules and essential synthetic biology skills for the ZQT-Nanjing high school team.

2. Established initial contact with the LU-NBBMS team from Jilin University to begin planning the collaborative illustration of the "Chassis Organism White Paper".

3. Attended the 3rd Synthetic Biology Conference & Exhibition (SBC), presenting our project and exploring potential collaborations with teams including Nanjing Agricultural University and Xi'an Jiaotong-Liverpool University.

1. Delivered iGEM rules and synthetic biology skills training to the ZQT-Nanjing high school team.

2. Held an online meeting with the iGEM team from Xi'an Jiaotong-Liverpool University (XJTLU) to discuss potential collaborations in sustainability and public education.

1. Consulted Professor Wang Xin, leading to the selection of the specialized high-lysine producing chassis strain E. coli NT1003.

2. Organized a "Synthetic Biology Carnival" on campus to introduce PA54 and green production concepts to students and faculty.

3. Participated in the Jiangsu-Zhejiang-Shanghai (JZS) iGEM Exchange, connecting with teams like Shanghai Jiao Tong University and Fudan University to share our project and gather feedback.

1. Sought advice on dynamic regulation switches from Professor Chen Kequan (recommended thermosensitive system) and Professor Liu Liming (recommended A-IDP encapsulation system).

2. Analyzed results from the public survey on bio-based materials to identify key focus areas for subsequent science communication.

3. Contributed to the writing and proofreading of specific sections for the collaborative "Chassis Organism White Paper" with Jilin University and 32 other teams.

1. Consulted Professor Xue Chuang to assess project feasibility and gather suggestions for metabolic pathway regulation.

2. Began designing community outreach lecture materials based on the analyzed survey data.

3. Continued collaborative writing and review process for the "Chassis Organism White Paper".

1. Consulted Professor Liu Guannan regarding leakage expression in the first-generation thermal switch; received advice to test promoters of different strengths.

2. Consulted Manager Guo Xiaojie from Tianmu Biotech regarding metabolic burden caused by high GAP enzyme expression; adopted the suggestion to switch to a low-copy-number plasmid.

3. Team members attended the 12th World Congress on Climate and Energy (WCCE), integrating insights from discussions with field experts into our project.

1. Visited the "Love Summer Tutoring Program" in Pukou District to deliver a synthetic biology lecture and conduct fun science demonstrations for children.

2. Attended the 11th Microbial Breeding Engineering and Application Evaluation Seminar, discussing potential collaborations and gathering valuable experimental suggestions from experts.

1. Consulted Professor Zhang Lei to address persistent leakage in the thermal switch system; implemented his suggestion to integrate RNA thermometers (U7/U8) for finer gene regulation.

1. Consulted Professor Liu Lixia on strategies for optimizing the key enzyme CadA, initiating a machine learning-assisted rational design approach.

2. Attended the 12th Conference of China iGEMer Community (CCiC), presented our project to experts, and received valuable optimization suggestions.

3. Published project updates and synthetic biology educational content on our social media platforms (WeChat Official Account and Xiaohongshu) to broaden our reach.

1. Communicated with expert Zhang Yutong from Nest.Bio to understand project cost control and explore commercial potential.

2. Collaborated with CJUH-JLU-China and 32 other teams to jointly release the "Synthetic Biology Rumors Debunking White Paper".

1. Interviewed leadership from China Textile Academy to understand challenges in PA54 industrialization and key considerations for optimizing lab protocols.

2. Discussed with Professor Wu Zhansheng from Xi'an Polytechnic University how to align synthetic biology research outcomes with industry regulations.

3. Interviewed experts from Xuan Kai Biotechnology to learn about critical factors for industrial scaling, including site selection and regulatory compliance.

4. After facing obstacles in modifying the CadA active site, consulted Dr. Chen Cai and pivoted our strategy to focus on predicting beneficial mutations that enhance decamer stability.

1. Consulted Professor Wei Guoguang for final project optimization advice, focusing on overall narrative, presentation logic, and visual improvements.

2. Finalized and officially released the first illustrated "Chassis Organism White Paper" as a core contribution to the iGEM community.