From June 8 to July 19, 2025, we established and optimized a biosynthetic system for 2′-fucosyllactose (2′-FL) production in E. coli. Initially, we introduced the manB and manC genes into BL21, enabling GDP-mannose synthesis and producing a baseline 2′-FL yield of ~8 mg/L. Subsequently, we constructed a complete biosynthetic pathway by assembling gmd, fcl, and futC into the same host, achieving a yield of ~20 mg/L after 72 h fermentation. To improve GDP-fucose availability, we further engineered a TrxA-futC fusion protein, which enhanced futC expression and solubility, boosting 2′-FL yield by ~56% to ~28 mg/L.

Extensive parameter optimization was performed, including variation of glycerol, yeast extract, lactose, and IPTG concentrations. Optimal conditions were identified as 20 g/L glycerol, 5 g/L yeast extract, 8 g/L lactose, and 0.5 mM IPTG. Comparative fermentation between BL21 and DH5α strains revealed that DH5α exhibited significantly higher productivity (158 mg/L vs. 8 mg/L), leading to its adoption as the preferred chassis.

A comprehensive 72-hour time-course experiment demonstrated consistent 2′-FL accumulation, lactose consumption, and biomass growth, verified by HPLC and enzymatic assays. Protein expression was validated by SDS-PAGE.

Finally, all data were consolidated and visualized using GraphPad Prism, validating the effectiveness of stepwise design strategies: from gene module construction to expression optimization and medium refinement. This robust System 1 provides a solid foundation for industrial 2′-FL biosynthesis.

📊 Weekly Gantt Chart

📅 Daily Experiment Schedule

Date	Experiment	Details	Notes
2025-06-08	Gene synthesis reception & LB media preparation	Received synthetic genes manB, manC, gmd, fcl, futC (codon-optimized, RFC#10 compliant); prepared LB agar plates & LB broth + Kana (100 µg/mL)	Plates stored inverted at 4 °C; genes aliquoted to avoid degradation
2025-06-09	Plasmid construction (pET28a-manB+manC)	Cloned manB+manC into pET28a using NdeI/XhoI; transformed into DH5α	Negative control included
2025-06-10	Colony PCR & sequencing of manB+manC	Screened 12 colonies; positive clones verified by sequencing	Sequencing confirmed correct insert
2025-06-11	BL21(DE3) transformation (manB+manC)	Transformed verified pET28a-manB+manC into BL21(DE3); prepared glycerol stocks	Stocks stored at −80 °C (25% glycerol)
2025-06-12	Fermentation setup (proof-of-concept)	Cultured BL21-manB+manC in M9 + glycerol + yeast extract; induced at OD600=0.6 with IPTG (0.5 mM) + lactose (8 g/L)	Sampling at 24, 48, 72 h
2025-06-13	2′-FL content assay (kit)	Collected 1 mL samples; centrifuged; supernatant stored at −20 °C; quantified via enzymatic cascade (α-L-fucosidase + FDH, A340)	BL21 control ≈ 0 mg/L; BL21-manB+manC ≈ 8.10 ± 1.03 mg/L
2025-06-14	Expansion to full pathway cloning	Assembled pET28a-(manB+manC+gmd+fcl+futC); transformed into DH5α	Colonies observed; sequencing submitted
2025-06-15	Colony PCR & sequencing (five-gene cassette)	Verified gmd, fcl, futC insertion; sequencing confirmed integrity	manA excluded after growth inhibition
2025-06-16	BL21(DE3) transformation (full pathway)	Introduced five-gene plasmid into BL21(DE3); glycerol stocks prepared	Robust growth observed
2025-06-17	Comparative fermentation (BL21 controls vs BL21-manB+manC vs BL21-manB+manC+gmd+fcl+futC)	Same M9 induction setup; sampling 0–72 h	Lactose and DCW also tracked
2025-06-18	Assays (2′-FL, lactose, DCW)	Kit analysis (2′-FL, lactose JL-T1072); oven-dried biomass for DCW	Final yields: 8.11 mg/L (2-gene) vs 19.66 ± 2.62 mg/L (5-gene); lactose consumed steadily; DCW ↑
2025-06-20	HPLC validation of fermentation products	Fermentation samples analyzed by WATERS ACQUITY UPLC BEH Amide, RID detection; retention time compared to 2′-FL standard	Peak at 4.25 min confirmed 2′-FL identity
2025-06-23	Chassis comparison (BL21 vs DH5α)	Constructed DH5α-five-gene strain; parallel fermentation under identical conditions	Yield: DH5α 158.26 ± 21.67 mg/L vs BL21 8.23 ± 1.86 mg/L (~19× higher)
2025-06-26	Protein engineering – TrxA fusion futC	Designed DH5α strain expressing TrxA-futC fusion; fermentation + 2′-FL assay	Yield: 279.47 ± 22.09 mg/L (+56% vs untagged futC)
2025-06-29	Replicates & statistical analysis	Performed ≥3 biological replicates for each strain; analyzed with GraphPad Prism (ANOVA, Tukey)	Significance p<0.0001 between strains
2025-07-01	Consolidated yield comparisons	Summarized multi-stage DBTL improvements: 2-gene (8.10 mg/L) → 5-gene (19.66 mg/L) → DH5α chassis (158.26 mg/L) → TrxA-futC (279.47 mg/L)	Clear 34× yield improvement across iterations
2025-07-05	Data validation & figure preparation	Compiled gel images, assay data, HPLC chromatograms, and yield bar plots for wiki	Figures cross-referenced to results section
2025-07-10	Instructor-guided review	Reviewed experimental flow, troubleshooting, and validated HPLC peak assignments	Confirmed orthogonal validation strategy
2025-07-15	Final statistical check & summary draft	Drafted results + conclusions for wiki system page; emphasized DBTL learnings	Incorporated significance annotations
2025-07-19	Reserved contingency day	Buffer slot for repeats if data loss/errors occur	Completed without major issues

From July 20 to August 2, 2025, we successfully developed and optimized a recombinant trypsin production system in E. coli BL21(DE3). The process began with the codon-optimized TRYP gene (porcine trypsinogen, UniProt: P00761), which was cloned into the pET-28a expression vector using NdeI and XhoI sites. The resulting plasmid was directly transformed into BL21(DE3), and correct transformants were confirmed by sequencing.

Protein expression was induced with IPTG, and SDS-PAGE analysis revealed a prominent ~25 kDa band corresponding to the trypsin zymogen. The expressed protein formed inclusion bodies, which were isolated, solubilized in 8 M urea, and subjected to a stepwise refolding protocol. Redox conditions were optimized (1 mM GSH / 0.1 mM GSSG), and proper folding was confirmed by solubility assessment and SDS-PAGE.

The refolded zymogen was activated using enterokinase to generate mature trypsin, and enzymatic activity was quantified using a BAPNA-based assay. The final product exhibited robust catalytic function, achieving ~75% activity relative to commercial trypsin standards.

This streamlined system—from gene construction and expression to purification, refolding, and activation—demonstrates high efficiency and reproducibility. All results were analyzed and visualized using GraphPad Prism, supporting the rational, stepwise design of System 2 and laying a strong foundation for future applications in protein digestion and industrial enzyme production.

📊 Weekly Gantt Chart

📅 Daily Experiment Schedule

Date	Experiment	Details	Notes
2025-07-20	Gene Reception & Vector Digestion	Received codon-optimized TRYP gene; pET-28a plasmid digested with NdeI/XhoI	TRYP gene synthesized per RFC10 standard; vector gel-purified post-digestion
2025-07-21	T4 Ligation	Ligated TRYP insert into linearized pET-28a vector	Reaction incubated overnight at 16°C; molar ratio optimized for insert:vector = 3:1
2025-07-22	Transformation into BL21(DE3)	Transformed ligation product into BL21(DE3); plated on LB + Kan (100 μg/mL)	Heat shock at 42°C; recovery in SOC; incubated at 37°C overnight
2025-07-23	Colony Selection & Miniprep	Picked colonies; cultured in LB + Kan; plasmid miniprep using commercial kit	Expected yield ~150–250 ng/μL; plasmids stored at -20°C
2025-07-24	Sequencing Verification	Verified TRYP insertion via Sanger sequencing	Correct clones selected for downstream protein expression
2025-07-25	Pre-culture & Protein Induction	Inoculated verified BL21-TRYP; induced with 0.5 mM IPTG at OD600 ≈ 0.6	Cultured at 25°C post-induction to promote inclusion body formation
2025-07-26	SDS-PAGE Analysis	Analyzed induced samples via SDS-PAGE; expected TRYP band ~25 kDa	Strong expression observed in induced sample; stored pellets at -80°C
2025-07-27	Inclusion Body Isolation	Cell lysis via sonication; centrifugation to collect inclusion body pellet	Pellet washed with Triton buffer and PBS; stored at -20°C
2025-07-28	Solubilization & Initial Refolding	Solubilized inclusion bodies in 8M urea; performed dialysis in decreasing urea concentrations	Refolding buffers included GSH/GSSG system; monitored protein solubility
2025-07-29	Refolding Optimization	Adjusted dialysis rate and redox conditions; analyzed refolding efficiency via SDS-PAGE	Best yield observed at 1 mM GSH / 0.1 mM GSSG; extended dialysis over 48 h
2025-07-30	Zymogen Activation	Activated refolded trypsinogen using enterokinase; monitored cleavage via SDS-PAGE	Clear conversion from zymogen to active form observed after 6 h incubation
2025-07-31	Trypsin Activity Assay Setup	Prepared BAPNA substrate; constructed p-nitroaniline standard curve at 405 nm	Ensured linear response range; positive control: commercial bovine trypsin
2025-08-01	Trypsin Activity Measurement	Assayed refolded trypsin activity; calculated specific activity in U/mg	Activity reached ~75% of commercial enzyme; low-activity batch retested
2025-08-02	Final Summary & Data Consolidation	Compiled data from all stages: cloning, expression, refolding, activation, assay	Figures and SDS-PAGE images saved for wiki; yield and activity data plotted and interpreted

From August 3 to August 15, 2025, we established and validated a recombinant lacZ expression system in E. coli for functional evaluation of β-galactosidase activity. We began by constructing a pET-28a-lacZ expression vector, using codon-optimized lacZ ORF and double digestion with NdeI/XhoI. Following T4 ligation and transformation into BL21, positive clones were screened via colony PCR and verified through Sanger sequencing.

The verified plasmid was transformed into BL21(DE3), and recombinant lacZ expression was induced using 1 mM IPTG at 18 °C. Successful overexpression was confirmed by Western blot, which detected a distinct band at ~116 kDa, corresponding to the full-length His-tagged lacZ protein.

To evaluate enzymatic function, the recombinant protein was purified via Ni-NTA affinity chromatography. A lactose degradation assay was established using 10 mM lactose as substrate, and glucose release was monitored over 8 hours using an Abcam Glucose Assay Kit. The results showed progressive lactose hydrolysis, reaching ~80% conversion, with glucose concentration increasing from 0.04 mM to 8.17 mM.

All data were processed using GraphPad Prism, confirming effective activity and expression of the engineered lacZ system. This work validates our System 3 design, from vector construction to functional protein analysis, laying a foundation for future applications in lactose metabolism and synthetic biology.

📊 Weekly Gantt Chart

📅 Daily Experiment Schedule

Date	Experiment	Details	Notes
2025-08-03	PCR Amplification of lacZ Gene	Amplified lacZ using gene-specific primers; ~3 kb expected band; product purified from gel	Verified size via agarose gel electrophoresis; minimized UV exposure
2025-08-04	Double Digestion of lacZ & Vector	lacZ and pET-28a(+) digested with EcoRI and HindIII	Insert and vector gel-purified and quantified
2025-08-05	Ligation & Transformation into BL21	T4 ligase used for overnight ligation; transformed into BL21 competent cells	Plated on LB-Kanamycin plates; negative control included
2025-08-06	Colony PCR Screening & Miniprep	Screened colonies using colony PCR; positive clones cultured overnight; plasmid extracted	8/10 colonies positive; plasmids prepared for sequencing
2025-08-07	Sequencing Verification of lacZ	Submitted positive plasmids for Sanger sequencing with T7/T7 terminator and internal primers	Sequences matched reference lacZ; no mutations
2025-08-08	Transformation into BL21(DE3)	Verified plasmid transformed into BL21(DE3); colonies selected and grown	Glycerol stocks prepared
2025-08-09	Protein Expression Induction (IPTG)	Cultured BL21-lacZ to OD600 ~0.6, induced with 1 mM IPTG; incubated at 18℃ overnight	Samples taken pre- and post-induction for SDS-PAGE
2025-08-10	SDS-PAGE & Western Blot	Performed SDS-PAGE and transferred proteins to PVDF membrane; probed with anti-His antibody	Detected ~116 kDa band consistent with lacZ; uninduced sample negative
2025-08-11	Protein Purification (Ni-NTA)	His-tagged lacZ purified via Ni-NTA column; elution fractions analyzed by SDS-PAGE	Major protein in elution fractions; concentration determined via Bradford assay
2025-08-12	Functional Assay Setup	Prepared ONPG (o-nitrophenyl-β-D-galactopyranoside) substrate assay to test β-galactosidase activity	Control (no enzyme) and heat-inactivated enzyme included
2025-08-13	Functional Assay Execution	Measured OD420 over time to quantify ONP (yellow product) formation	Active enzyme showed clear increase; heat-inactivated and control minimal activity
2025-08-14	Data Analysis & Graph Generation	Processed ONPG assay data; plotted time vs OD420 to assess enzyme kinetics	Calculated Vmax and Km using Michaelis-Menten model
2025-08-15	Summary & Verification Report	Compiled all data: construct verification, expression analysis, functional assay	Final report drafted; included images of gels, blots, graphs; confirmed successful expression and functionality of recombinant lacZ protein