iDEC 2025 Project Notebook: Gut Health Guardian
November 2024: Project Initiation and In-Depth Research
This month, the team was officially formed and identified "perioperative gut microbiota dysbiosis" as the core problem. We conducted intensive literature review, focusing on the impact of surgical procedures and antibiotic use on gut microecology, as well as the limitations of existing probiotic therapies. Based on this, we held multiple online seminars and preliminarily established the engineering bacteria design concept using Lactobacillus casei as the chassis, integrating the three core modules of "sensing-treatment-safety". Lesson Learned: Thorough preliminary research is the cornerstone of project success, helping us precisely define clinical needs and technological gaps, avoiding blindness in subsequent work.
December 2024: Molecular Design and Part Selection
The focus shifted to specific molecular design. We obtained sequences for accA, OLE1, DEFB4A, and the promoter P16090 from the NCBI database and performed full-gene codon optimization on OLE1 based on the codon preference of Lactobacillus. Simultaneously, we began drafting detailed experimental protocols and contacted gene synthesis companies to initiate the synthesis of core components. Lesson Learned: Early codon optimization and sequence synthesis can significantly shorten subsequent construction cycles; detailed experimental protocols ensure efficient collaboration.
January 2025: Chassis Cell Validation and Plasmid Backbone Preparation
We first conducted bile tolerance tests on wild-type L. casei, confirming its normal growth in LB solid and liquid media containing up to 0.5% bile salts, laying the foundation for the subsequent use of the bile-responsive promoter. Concurrently, we successfully extracted plasmid backbones like pMG36e and PNZ8048 and began restriction enzyme digestion verification in preparation for gene cloning.
February 2025: Basic Plasmid Construction and Transformation
The core task this month was constructing the first batch of plasmids. Using restriction enzyme digestion and ligation, we successfully built the constitutive expression plasmid Psod-accA/AmpR and transformed it into L. casei competent cells via heat shock. Positive clones were selected on ampicillin-containing plates and preliminarily verified through colony PCR and plasmid digestion identification. Lesson Learned: The efficiency of competent cell preparation is crucial for successful transformation and requires strict optimization of the preparation protocol.
March 2025: Functional Validation of the Bile Salt Response System
We successfully constructed the reporter plasmid P16090-GFP/KanR and transformed it into the chassis cells. Culturing the engineered bacteria in media with different bile salt concentrations and using fluorescence microscopy and microplate reader quantification confirmed that the P16090 promoter could be specifically induced by bile salts, with fluorescence intensity positively correlating with bile salt concentration. This result indicated our core regulatory switch functioned normally.
April 2025: Oleic Acid Synthesis Module Integration and Testing
After validating the promoter function, we constructed the therapeutic plasmid P16090-OLE1/KanR. After culturing the transformants, the OLE1 protein with a His-tag was detected under induced conditions via Western Blot. More importantly, analysis of the culture supernatant using High-Performance Liquid Chromatography confirmed a significant increase in oleic acid production compared to the wild-type strain. Lesson Learned: Detecting metabolites requires meticulous sample preparation to exclude interference from the medium background.
May 2025: Defensin Module Integration and Co-expression
To achieve multiple therapeutic functions, we designed a fusion expression unit, linking OLE1 and DEFB4A via a self-cleaving 2A peptide, constructing the plasmid P16090-OLE1-2A-DEFB4A. Western Blot results showed both protein bands were correctly expressed. We further established an L-929 cell inflammation model and found the engineered bacterial supernatant effectively alleviated cellular oxidative stress, preliminarily demonstrating its anti-inflammatory activity.
June 2025: Biosafety System Construction and In Vitro Validation
This month focused on building the biocontainment system. We successfully assembled preca-Hok and P16090-Sok into the stringent plasmid PIP501. Subsequently, we performed a time-kill curve assay on engineered bacteria carrying this system, finding that viable bacterial counts dropped by over 4 orders of magnitude within 8 hours after bile salt removal, strongly proving the efficiency and reliability of the Hok/Sok system.
July 2025: Comprehensive Functional Validation and Mid-Term Exchange
We began integrating the therapeutic and safety modules into the same engineered bacterial strain and conducted comprehensive functional re-verification. Team representatives attended the CCIC\(Conference of China iDECer Community\), presented our mid-term results as a poster, and engaged in in-depth discussions with field experts, gaining valuable feedback. Lesson Learned: External exchanges are excellent opportunities to test project innovation and gain inspiration.
August 2025: Data Supplementation and Human Practices Expansion
Addressing questions raised by experts during exchanges, we supplemented data with co-culture competition experiments between our engineered bacteria and common intestinal pathogens, further validating the bacteriostatic function of defensin. Meanwhile, members of the Human Practices team visited local hospital gastrointestinal surgery departments, interviewed medical staff and postoperative patients, deepened their understanding of clinical needs, and collected firsthand feedback for our solution.
September 2025: Data Consolidation, Modeling, and Wiki Finalization
Wet lab work essentially concluded, entering the data consolidation phase. We aggregated all experimental results, performed statistical analysis, and produced final charts and graphs. Concurrently, we developed a simple kinetic model based on ordinary differential equations to simulate the colonization and clearance dynamics of our engineered bacteria in the gut. The team's core task was the design and development of the Wiki page, ensuring all content was written, programmed, and polished before the deadline.
October 2025: Final Preparation and iDEC Giant Jamboree
This was the final sprint. We focused intensely on refining the Final Presentation slides and Poster, conducting multiple rehearsal sessions. The project promotion video was also finalized and released. This month, the entire team traveled to Paris, France, to participate in the iDEC Giant Jamboree, presenting our "Gut Health Guardian" project to the global synthetic biology community, exchanging ideas with teams from around the world, and drawing a successful conclusion to a full year of hard work.