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Cycle 1 – Bacterial Cellulose (BC) Production System (06/16–07/08)


From June 16 to July 8, 2025, we successfully established and characterized a system for producing the structural biopolymer, bacterial cellulose (BC), in E. coli BL21(DE3). We constructed the core production system by introducing a dual-gene cassette containing codon-optimizedbcsA and bcsB from Gluconacetobacter xylinus into the pSB1A3 plasmid, driven by a strong constitutive promoter. Comparative analysis against a control strain (carrying an empty vector) confirmed the system's efficacy; the engineered strain demonstrated significantly higher BC yield (p < 0.05) via dry weight analysis and superior per-cell BC production as validated by a Congo Red binding assay (p < 0.0001). A comprehensive 60-hour time-course experiment was conducted to characterize production kinetics, revealing that BC accumulation enters a plateau phase at approximately 48 hours. This study established 48 hours as the optimal fermentation period and achieved a peak yield of approximately 970 mg/L. The successful construction of the genetic circuit was verified by colony PCR and agarose gel electrophoresis. All data were analyzed to validate the effectiveness of the design strategy. This robust "version 1.0" BC production system provides a critical performance baseline and serves as the foundational structural backbone for the ReGenStitch project.


Daily Experiment Schedule

Date Experiment Title Experiment Objective Key Steps Main Results Notes
2025-06-16 Cycle 1 — Kickoff & Safety SOPs Align DBTL plan and confirm BSL-1 practices Team briefing; waste/sterility SOPs; assign roles; reagent inventory Project plan approved; safety roles assigned Final product is cell-free biomaterial; BSL-1 rules logged.
2025-06-18 Cycle 1 — bcsAB Design Freeze Lock design for E. coli bcsA/bcsB expression Confirm RFC10 sites; J23100+B0034 parts; primer list finalized Design pack completed Cycle-1 goal: BC production in E. coli BL21(DE3).
2025-06-20 Cycle 1 — Media & Competent Cells Prepare plates/media and cells for cloning Pour LB-Amp plates (invert, 4 °C); prep DH5α/BL21(DE3) competent cells Plates ready; aliquoted competent cells Media/antibiotic usage per cloning steps.
2025-06-22 Cycle 1 — PCR of bcsAB Inserts Obtain clean bcsAB fragments Gradient PCR; gel; gel extraction; quantify Clear bands at expected size Store inserts on ice → −20 °C; minimize UV time.
2025-06-24 Cycle 1 — pSB1A3 Digest & Ligation (bcsAB) Assemble bcsAB into pSB1A3 Double digest pSB1A3; T4 ligation (insert:vector ratios); include vector-only control Ligation reactions complete Standard BioBrick/RFC10-style build.
2025-06-26 Cycle 1 — DH5α Transformation (bcsAB) Introduce ligation products for plasmid amplification Heat-shock TF; SOC recovery; plate on LB-Amp Colonies on ligation plates Negative control plate clean (good).
2025-06-28 Cycle 1 — Colony PCR Screen (bcsAB) Identify correct recombinant clones Pick 12 colonies; colony PCR across junctions; choose positives 8/12 positive clones Start overnight cultures for positives.
2025-06-30 Cycle 1 — Miniprep & Sanger Submit (bcsAB) Purify plasmids and submit for sequencing Miniprep; NanoDrop; prepare VF2/VR + internal primers; ship 8 plasmids (60–180 ng/µL) submitted Sequence verification per plan.
2025-07-02 Cycle 1 — Wait: Sequencing in Progress Hold wet work; pre-plan expression tests Draft BC culture conditions; label BL21 plates; update DBTL tracker — DBTL loop documented for Cycle-1.
2025-07-04 Cycle 1 — Sequence Review & BL21(DE3) TF Select error-free bcsAB plasmid(s) and move to host Review chromatograms; transform BL21(DE3); make glycerol stocks 2 perfect clones; robust BL21 colonies BL21 (DE3) chosen as Cycle-1 chassis.
2025-07-06 Cycle 1 — BC Pilot Production (Static Culture) Validate bacterial cellulose production Inoculate glucose-rich static bottles (28–30 °C) with bcsAB strain + EV control Pellicles visible in 24–48 h Congo red binding planned as identity check.
2025-07-08 Cycle 1 — BC Harvest & Basic QC Harvest and quantify BC Alkali wash; neutralize; dry mass (mg/L); image pellicles Measurable BC yield; consistent morphology Record bottle fill volume and area for normalization.


Cycle 2 – Reusable Whole-Cell Biocatalyst System (07/10–07/30)


From July 10 to July 30, 2025, we established and characterized a reusable whole-cell biocatalyst for the production of antimicrobial Chitosan Oligosaccharides (COS). We engineered a cell surface display system in E. coli by fusing the chitosanase CHI1 to the N-terminus of the Ice Nucleation Protein (INP) anchor. A head-to-head performance test demonstrated that our whole-cell biocatalyst was significantly more efficient (p < 0.01) in both COS production and enzyme activity compared to traditional crude enzyme lysate. Further characterization identified the optimal operating conditions as 50°C and pH 7.0. To validate its real-world applicability, we demonstrated an end-to-end sustainable process: first, we achieved a high chitin yield of ~14.5% from shrimp shell waste using a co-fermentation of B. subtilis and Acetobacter sp.; second, our whole-cell catalyst showed superior hydrolysis efficiency on this crude, sustainably-sourced substrate. The successful construction of the INP-CHI1 fusion gene was verified by colony PCR. This cycle successfully developed a highly efficient, low-cost, and reusable biocatalyst, providing the core antimicrobial and healing-promoting module for the ReGenStitch project.


Daily Experiment Schedule

Date Experiment Title Experiment Objective Key Steps Main Results Notes
2025-07-10 Cycle 2 — INP-CHI Plan & Primer Check Prepare surface-display chitosanase build Confirm INP anchor fusion design; finalize primers and linkers Design validated Cycle-2 goal: reusable whole-cell catalyst.
2025-07-12 Cycle 2 — PCR CHI1 & Backbone Prep Generate CHI1 insert and prepare vector PCR CHI1; gel-purify; digest pSB1A3-INP backbone Clean insert and vector on hand Proceed to ligation next day.
2025-07-14 Cycle 2 — Ligation & DH5α TF (INP-CHI) Clone INP-CHI into plasmid backbone T4 ligation; transform DH5α; plate on LB-Amp; include no-insert control Colonies obtained Track insert:vector molar ratio.
2025-07-16 Cycle 2 — Colony PCR & Overnights (INP-CHI) Identify correct assembly clones Screen 12 colonies; select positives; start ON cultures 9/12 positives Prepare glycerols of positives.
2025-07-18 Cycle 2 — Miniprep & Sanger Submit (INP-CHI) Verify INP-CHI sequence Miniprep; quantify; submit VF2/VR + internal Samples submitted Maintain both display and intracellular CHI variants.
2025-07-20 Cycle 2 — Wait: Sequencing/Logistics Bridge time for reads & consumables Order standardized chitosan substrate; prepare buffers — Plate reader and baseline blanks prepared.
2025-07-22 Cycle 2 — BL21(DE3) TF (INP-CHI) Create expression strain(s) Transform BL21(DE3); select colonies; prepare glycerol stocks BL21-INP-CHI established Keep EV and intracellular-CHI controls.
2025-07-24 Cycle 2 — Expression Induction & Whole-Cell Prep Confirm surface display and prepare catalysts Induce at mid-log; gentle handling; prepare washed whole-cells Good biomass; intact prep Avoid harsh lysis to preserve display.
2025-07-26 Cycle 2 — CHI Activity & Benchmark vs Crude Demonstrate catalytic activity vs crude-enzyme approach Incubate with chitosan substrate; measure released products vs controls Displayed CHI > crude enzyme Matches reported superiority of whole-cell catalyst.
2025-07-28 Cycle 2 — pH/Temperature Optimization Identify optimal operating conditions Test pH 6–8 and 30–55 °C; fixed time; replicate Best activity near pH 7, ~50 °C Conditions align with prior results.
2025-07-30 Cycle 2 — Reusability (Rounds 1–3) Evaluate recyclability of whole-cell catalyst Reuse same biomass across runs; wash between cycles Activity retained over cycles Confirms “reusable” catalyst concept.

Cycle 3 – Curcumin Biosynthetic Pathway System (08/01–08/23)


From August 1 to August 23, 2025, we established and characterized a biosynthetic pathway for the anti-inflammatory agent curcumin in E. coli. We successfully reconstructed the plant-derived pathway by co-expressing the codon-optimized genes DCS and CURS1 in E. coli BL21(DE3) using an IPTG-inducible pET expression system. The successful construction of the plasmid was verified by colony PCR. An in vitro enzymatic assay using crude enzyme lysate demonstrated the de novo synthesis of curcumin, achieving a yield of approximately 13-14 μM, which was over 26 times higher than the control (p < 0.001). Further enzyme kinetics characterization yielded a Vmax of approximately 345.6 nM/min and a Km of 114.6 μM, confirming the system's catalytic efficiency. This cycle successfully validated the final core functional module—anti-inflammatory and scar regulation—for the ReGenStitch project.


Daily Experiment Schedule

Date Experiment Title Experiment Objective Key Steps Main Results Notes
2025-08-01 Cycle 3 — DCS/CURS Pathway Plan Prepare anti-inflammatory module build Confirm DCS + CURS1 genes and expression design in E. coli BL21 Build plan approved Cycle-3 goal: curcumin biosynthesis.
2025-08-03 Cycle 3 — PCR DCS/CURS & DH5α Assembly Generate inserts and assemble pathway vector PCR DCS/CURS; gel-purify; ligation into expression backbone; transform DH5α Colonies observed Proceed to screening and sequence-check next.
2025-08-05 Cycle 3 — Colony PCR Screen (DCS/CURS1) Identify correct DH5α clones Screen 12 colonies across junctions; select positives for overnight culture 7/12 positives at expected sizes Proceed to miniprep & Sanger.
2025-08-07 Cycle 3 — Miniprep & Sanger Submit Sequence-verify pathway Miniprep; quantify; submit VF2/VR + internal primers Plasmids sent for sequencing Track any mixed peaks; hold backups.
2025-08-09 Cycle 3 — Wait: Sequencing in Progress Hold benchwork pending reads Prepare BL21(Kan) plates; outline induction conditions and sampling plan — DBTL notes updated for Cycle-3.
2025-08-11 Cycle 3 — Sequence Review & BL21(DE3) Transformation Select error-free clone(s), move to expression host Inspect chromatograms; transform BL21(DE3); prepare glycerol stocks Robust transformants obtained Keep EV BL21 control.
2025-08-13 Cycle 3 — Induction Pilot & Lysate Prep Produce active DCS/CURS1 enzymes IPTG induction at mid-log; harvest; crude lysate & clarification Protein-rich lysate prepared Aliquot & store at −80 °C.
2025-08-15 Cycle 3 — Curcumin Assay (UV–Vis @420 nm) Demonstrate pathway activity Reaction with substrates; extract; measure A420 vs standards Clear 420 nm peak above controls Confirms anti-inflammatory module function.
2025-08-17 Cycle 3 — Kinetics/Optimization Estimate performance parameters Substrate gradient; time-course; fit initial rates Vmax/Km trends consistent with expectations Choose best induction temperature/time.
2025-08-19 Cycle 3 — Troubleshooting/Media Tuning Improve yields if suboptimal Adjust precursors/cofactors and pH per plan; re-assay Improved yield vs baseline Lock selected conditions.
2025-08-21 Cycle 3 — Module Freeze & Documentation Finalize constructs and SOPs Freeze working glycerols; finalize protocols and raw-data sheets Cycle-3 module closed Ready for integration.
2025-08-23 Cycle 3 — Buffer/Contingency Reserve for repeats or logistics Use only if sequencing/shipping delays occur; otherwise maintain readiness — Maintains schedule flexibility.
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Cycle 4 – System Integration and Suture Prototyping (08/25–09/17)


From August 25 to September 17, 2025, we completed the system integration phase by designing and fabricating the first physical prototype of the ReGenStitch suture. We integrated the bioactive components from previous cycles—bacterial cellulose (BC), chitosan (CS), and curcumin—using a solution blending, casting, and slitting method, with glycerol added as a plasticizer to ensure flexibility. The prototype's performance was validated through macroscopic evaluation against commercial sutures and a simulated suturing test on pigskin. The results confirmed that our prototype possesses sufficient mechanical strength to penetrate tissue and good flexibility for secure knotting. This cycle successfully transitioned the project from individual biological modules to a tangible device, culminating in a 'proof-of-concept' ReGenStitch v1.0 prototype and validating the feasibility of our integrated design.


Daily Experiment Schedule

Date Experiment Title Experiment Objective Key Steps Main Results Notes
2025-08-25 Cycle 4 — Material Prep (BC & Chitosan) Prepare inputs for composite Harvest/alkali-wash BC; neutralize; prepare chitosan solution Clean BC mats; clear CS solution Inputs ready for blending.
2025-08-27 Cycle 4 — Solution Blending (v1) Create first BC/CS composite dope Mix BC + chitosan; add plasticizer per plan; degas Homogeneous blend achieved Record exact ratios for DBTL.
2025-08-29 Cycle 4 — Casting & Controlled Drying (v1) Produce cast films for thread-making Level plates; controlled drying; peel intact films Intact films formed Label thickness and drying time.
2025-08-31 Cycle 4 — Slicing into Threads & Conditioning Convert films to line-like strips Precision-cut; rinse/condition; dry under tension Threads of consistent gauge Photo & gauge each set.
2025-09-02 Cycle 4 — Handling Check & Storage Prep Confirm basic handling integrity Gentle flex/knot handling; package dry samples Threads intact after handling Store in clean, dry pouches.
2025-09-04 Cycle 4 — Mechanical Handling & Knotting Test (v1) Assess strength & operability Standardized hand-knot pulls and handling observations Meets basic handling expectations Record force/extension qualitatively.
2025-09-06 Cycle 4 — CHI Activity on Composite Threads Check CHI functional retention Prepare whole-thread samples; run activity readout vs controls Detectable activity retained Aligns with Cycle-2 CHI findings.
2025-09-08 Cycle 4 — DBTL “Learn” Review Decide formulation updates Analyze v1 handling + activity; select improved ratios for v2 v2 plan approved Document changes for traceability.
2025-09-10 Cycle 4 — Formulation v2 Casting & Threading Produce refined prototype Blend per v2; cast; slice; condition as before v2 threads produced Label sets for demo.
2025-09-12 Cycle 4 — Pig-Skin Suturing Demo (v2) Validate operability on tissue surrogate Side-by-side suturing on pork skin vs commercial Smooth passage; secure knots Demonstrates practical handling.
2025-09-14 Cycle 4 — Imaging & Documentation Consolidation Capture evidence and organize data Photograph threads/demos; compile lab notes & figures Complete photo/data set Pre-final wiki/Notebook collation.
2025-09-15 Cycle 4 — Prototype Packaging & Labeling Prepare demonstration sets Label batches; store dry; document lot info Prototypes packaged Ready for final checks.
2025-09-16 Cycle 4 — Safety Review & Final Checks Reconfirm BSL-1 & cell-free deliverable Verify records; waste logs; confirm no live GMO in product Safety dossier complete Matches project safety design.
2025-09-17 Cycle 4 — Data Lock & Gantt Mapping Freeze deliverables and map cycles Finalize datasets, figures, SOPs; tag items by Cycle 1–4 for Gantt Project book closed Ready for wiki/presentation.
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