iGEM 2025 — Team sr-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

Content

* [System 1 – 2′-Fucosyllactose Production System (06/08–07/19)](#section-1)
  + [📊 Weekly Gantt Chart](#subsection-2)
  + [📅 Daily Experiment Schedule](#subsection-3)
* [System 2 – Trypsin Production System (07/20–08/02)](#section-4)
  + [📊 Weekly Gantt Chart](#subsection-5)
  + [📅 Daily Experiment Schedule](#subsection-6)
* [System 3 – LacZ Production System (08/03–08/15)](#section-7)
  + [📊 Weekly Gantt Chart](#subsection-8)
  + [📅 Daily Experiment Schedule](#subsection-9)

# System 1 – 2′-Fucosyllactose Production System (06/08–07/19)

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

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/5986/notebook/img1.webp)

## 📅 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 |

system1-notebook-2.pdf

2.2MB

[Download](https://static.igem.wiki/teams/5986/notebook/system1-notebook-2.pdf)

# System 2 – Trypsin Production System (07/20–08/02)

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

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/5986/notebook/img2.webp)

## 📅 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 |

System 2-notebook-2.pdf

1.4MB

[Download](https://static.igem.wiki/teams/5986/notebook/system-2-notebook-2.pdf)

# System 3 – LacZ Production System (08/03–08/15)

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

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/5986/notebook/img3.webp)

## 📅 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 |

System3-notebook-2.pdf

1.4MB

[Download](https://static.igem.wiki/teams/5986/notebook/system3-notebook-2.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Content

* [Engineering Success](#section-1)
  + [System 1: De Novo Biosynthesis of 2'-Fucosyllactose (2'-FL)](#section-2)
    - [Cycle 1.1: Proof-of-Concept with a Minimal Pathway (*manB+manC*)](#subsection-3)
    - [Cycle 1.2: Pathway Completion and Full Pathway Validation](#subsection-4)
    - [Cycle 1.3: Chassis Optimization to Eliminate Substrate Competition](#subsection-5)
    - [Cycle 1.4: Enhancing Key Enzyme Solubility with a Fusion Tag](#subsection-6)
  + [System 2: Production of Functional Recombinant Trypsin](#section-7)
    - [System 3: Functional Characterization of β-Galactosidase (*lacZ*)](#section-8)
      * [Reference](#section-9)

# Engineering Success

Our project, "ProBabyotics," is founded on a systematic and iterative engineering strategy. We aimed to develop a modular platform to enhance infant nutrition by producing three key functional biomolecules: the human milk oligosaccharide 2'-fucosyllactose (2'-FL) for immune support, the protease trypsin for improved protein digestibility, and the enzyme β-galactosidase to combat lactose intolerance.

We rigorously applied the Design-Build-Test-Learn (DBTL) cycle to tackle the challenges inherent in each module. For our most complex system, the de novo biosynthesis of 2'-FL, we executed a four-stage engineering cycle that progressively identified and solved key bottlenecks. This iterative process, which involved pathway completion, chassis optimization, and targeted protein engineering, resulted in a more than 34-fold increase in final product titer, from an initial 8.10 mg/L to a final 279.47 mg/L. For our other two modules, we successfully demonstrated complete "gene-to-function" pipelines, validating the expression and high catalytic activity of our engineered enzymes.

![Engineering Success Diagram - Overview](https://static.igem.wiki/teams/5986/engineering/img1.webp)

Click for detailed version

![Engineering Success Diagram - Detailed](https://static.igem.wiki/teams/5986/engineering/img2.webp)

Figure 1: Abstract figure for the three DBTL systems

This page documents our engineering journey, demonstrating how each DBTL cycle provided critical data and insights that informed the design of the subsequent iteration, leading to a robust and successful outcome for all three project goals.

# System 1: De Novo Biosynthesis of 2'-Fucosyllactose (2'-FL)

Our primary engineering goal was to create a highly efficient microbial cell factory for the low-cost production of 2'-FL. We achieved this through a four-part DBTL cycle.

## Cycle 1.1: Proof-of-Concept with a Minimal Pathway (*manB+manC*)

### Design

Our initial design was to establish a proof-of-concept for the 2'-FL biosynthetic pathway. We hypothesized that the first two enzymes, phosphomannomutase (*manB*) and mannose-1-phosphate guanylyltransferase (*manC*), would be sufficient to initiate the synthesis of 2'-FL by converting endogenous precursors and exogenous lactose(Li et al., 2018). We designed a minimal genetic circuit containing the *manB* and *manC* genes, cloned into a pET28a vector under the control of an inducible T7 promoter.

![Cycle 1.1: Proof-of-Concept with a Minimal Pathway (<em>manB+manC</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img3.webp)

Figure 2: Construction of BL21-*manB+manC*. (A) The plasmid map of pET28a-*manB+manC*. (B) The gene circuit of BL21-*manB+manC*.

### Build

The *manB* and *manC* genes were synthesized and cloned into the pET28a vector. The resulting plasmid was transformed into *E. coli* BL21(DE3). Successful construction of the recombinant plasmid was confirmed by agarose gel electrophoresis, which showed a distinct band at the expected size of 1947 bp(Figure 3).

![Cycle 1.1: Proof-of-Concept with a Minimal Pathway (<em>manB+manC</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img4.webp)

Figure 3: (A)Flow chart of bacterial culture; (B)the agarose gel electrophoresis analysis of the *manB+manC* gene fragment

Agarose gel electrophoresis analysis confirming the successful cloning of the *manB*+*manC* gene fragment (1947 bp).

### Test

The engineered BL21-*manB*+*manC* strain was cultured in M9 minimal medium with glycerol and yeast extract. Production was induced with 0.5 mM IPTG and 8 g/L lactose. After 72 hours of fermentation, the 2'-FL concentration in the supernatant was quantified using a commercial detection kit. The engineered strain was compared against control strains (wild-type BL21 and BL21 with an empty pET28a vector)(Figure 4, A).

![Cycle 1.1: Proof-of-Concept with a Minimal Pathway (<em>manB+manC</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img5.webp)

Figure 4: Comparison of 2'-FL yield in BL21-*manB+manC* after 72-hour fermentation

The BL21-*manB*+*manC* strain produced 8.10 ± 1.03 mg/L, while control strains showed negligible production(Figure 4, B).

### Learn

The results provided a crucial proof-of-concept. The co-expression of *manB* and *manC* was sufficient to initiate 2'-FL synthesis. However, the modest yield strongly indicated that the downstream steps—the synthesis of the GDP-L-fucose donor and the final fucosylation reaction—were the primary metabolic bottleneck(Byun et al., 2006). This learning directly informed our next design iteration: to construct the complete synthesis pathway.

## Cycle 1.2: Pathway Completion and Full Pathway Validation

### Design

Based on the learnings from Cycle 1.1, we designed a complete de novo synthesis pathway. We hypothesized that adding the three downstream genes—GDP-D-mannose-4,6-dehydratase (*gmd*), GDP-L-fucose synthase (*fcl*), and α-1,2-fucosyltransferase (*futC*)—would overcome the previously identified bottleneck and significantly increase the 2'-FL yield(Bonin

et al., 1997). The new composite part (Part: BBa\_25DDSHWK) integrates all five genes into a single operon under the control of the T7 promoter(Figure 5).

![Cycle 1.2: Pathway Completion and Full Pathway Validation Diagram](https://static.igem.wiki/teams/5986/engineering/img6.webp)

Figure 5: Construction of BL21-*manB+manC*+*gmd*+*fcl*+*futC*. (A) The plasmid map of pET28a-*manB+manC*+*gmd*+*fcl*+*futC* (B) The gene circuit of BL21-*manB+manC*+*gmd*+*fcl*+*futC*.

### Build

The *gmd*, *fcl*, and *futC* genes were sequentially cloned into the pET28a-*manB*+*manC* plasmid, creating the final five-gene construct. The plasmid was then transformed into *E. coli* BL21(DE3). The successful integration of the new gene fragments was confirmed by agarose gel electrophoresis, which showed distinct bands at the expected sizes for *gmd* (1119 bp), *fcl* (963 bp), and *futC*(1095 bp)(Figure 6).

![Cycle 1.2: Pathway Completion and Full Pathway Validation Diagram](https://static.igem.wiki/teams/5986/engineering/img7-new.avif)

Figure 6: (A)Flow chart of bacterial culture; (B) Agarose gel electrophoresis analysis confirming the successful cloning of the *gmd*, *fcl*, and *futC* gene fragments.

### Test

With the full pathway constructed, we conducted a comprehensive series of tests to quantify yield, verify product identity, and understand the system's dynamics.

Yield Enhancement: We first performed a comparative fermentation experiment under the same conditions as Cycle 1.1. The new five-gene strain (BL21-*manB*+*manC*+*gmd*+*fcl*+*futC*) showed a final yield of 19.66 ± 2.62 mg/L, a significant 2.4-fold increase over the 8.11 ± 1.04 mg/L produced by the initial two-gene strain. This confirmed that completing the pathway dramatically boosted production(Figure 6).

![Cycle 1.2: Pathway Completion and Full Pathway Validation Diagram](https://static.igem.wiki/teams/5986/engineering/img8.webp)

Figure 6: (A)Flow chart of 2'-FL content assay; (B)Yield comparison of 2 '-fucosyllactose after 72 hours of fermentation of BL21 recombinant strain

The strain with the complete pathway (BL21-*manB*+*manC+gmd+fcl+futC*) produced significantly more 2'-FL than the initial strain (BL21*-manB*+*manC*) and controls(Figure 6).

Product Identity Verification by HPLC: To unambiguously confirm that our engineered strain was producing the correct molecule, we analyzed the fermentation broth using High-Performance Liquid Chromatography (HPLC). The chromatogram revealed a prominent peak at a retention time of 4.25 minutes, which perfectly matched the retention time of a pure 2'-FL analytical standard run under identical conditions. This result provided definitive evidence that our system was successfully synthesizing 2'-FL(Figure 7).

![Cycle 1.2: Pathway Completion and Full Pathway Validation Diagram](https://static.igem.wiki/teams/5986/engineering/img9.webp)

Figure 7: (A)Mechanism of HPLC; (B)HPLC analysis of fermentation products of BL21.

Fermentation Dynamics: To understand the relationship between substrate consumption and product formation, we conducted a 72-hour dynamic fermentation study. We monitored cell growth (Dry Cell Weight), lactose concentration, and 2'-FL concentration over time. The results showed a clear inverse correlation: as the cells grew, the lactose concentration in the medium steadily decreased, while the concentration of 2'-FL consistently increased. This confirmed that our engineered pathway was actively converting the lactose substrate into the target product(Figure 8).

![Cycle 1.2: Pathway Completion and Full Pathway Validation Diagram](https://static.igem.wiki/teams/5986/engineering/img10.webp)

Figure 8: (A)(B)(C)Experimental Flowchart; (D) Changes in 2'-FL yield, Lactose Content, and Cell Dry Weight Over Time in BL21-*manB+manC*+*gmd*+*fcl*+*futC.*

### Learn

This round of experiments successfully transformed our proof-of-concept into a fully functional and validated production pathway. We gained three critical insights:

Pathway completion is essential: The 2.4-fold yield increase proved that the downstream enzymes were a primary bottleneck.

The product is authentic: HPLC analysis provided definitive confirmation that our system was producing genuine 2'-FL, validating all our quantitative measurements.

The system functions as designed: The dynamic fermentation data showed a clear link between lactose consumption and 2'-FL production.

With the product identity confirmed and its synthesis from lactose established, our analysis shifted towards metabolic efficiency. The dynamic fermentation data showed continuous lactose consumption, prompting the question: was all the lactose being used for 2'-FL synthesis? We hypothesized that a significant portion was being diverted by the host's native metabolic pathways. This led us to identify the *lacZ* gene in the *E. coli* BL21 chassis as a prime suspect for creating a competitive pathway that hydrolyzes our key substrate. This insight directly informed our next design cycle: to optimize the host chassis by eliminating this substrate competition.

## Cycle 1.3: Chassis Optimization to Eliminate Substrate Competition

### Design

In analyzing the metabolism of our *E. coli* BL21(DE3) chassis, we identified a critical flaw: the native β-galactosidase enzyme, encoded by the *lacZ* gene, directly competes for our lactose substrate by hydrolyzing it into glucose and galactose(chin et al., 2021). We hypothesized that this substrate competition was a major bottleneck. To test this, we designed an experiment to compare our system's performance in BL21 versus *E. coli* DH5α, a common lab strain that is naturally *la**cZ*-deficient(Figure 9).

![Cycle 1.3: Chassis Optimization to Eliminate Substrate Competition Diagram](https://static.igem.wiki/teams/5986/engineering/img11.webp)

Figure 9: Methods for Exploring the effect of two different strains(BL21/DH5α) on 2'FL yield. (A)Experimental Methods; (B)Experimental Flowchart

### Build

The pET28a-*manB*+*manC*-*gmd*-*fcl*-*futC* plasmid containing our complete five-gene operon was transformed into chemically competent *E. coli* DH5α cells, creating a new engineered strain for a direct head-to-head comparison.

### Test

We conducted a comparative fermentation experiment, culturing both the DH5α-based strain and the original BL21-based strain under identical conditions. After 72 hours, the final 2'-FL concentration was quantified.

![Cycle 1.3: Chassis Optimization to Eliminate Substrate Competition Diagram](https://static.igem.wiki/teams/5986/engineering/img12.webp)

Figure 10: Comparison of 2'-FL production in BL21 vs. DH5α chassis.

The *lacZ*-deficient DH5α strain produced 158.26 ± 21.67 mg/L, a ~19-fold improvement over the 8.23 ± 1.86 mg/L from the BL21 strain(Figure 10).

### Learn

The dramatic ~19-fold increase in yield powerfully validated our hypothesis. Substrate competition from the native *lacZ* gene was a major limiting factor. By simply switching to a more suitable chassis, we successfully redirected metabolic flux towards our product. This critical learning established DH5α as our optimized chassis for all subsequent work and highlighted the importance of host selection in metabolic engineering.

## Cycle 1.4: Enhancing Key Enzyme Solubility with a Fusion Tag

### Design

Having optimized the chassis, we focused on the final rate-limiting enzyme, *futC*, which is of human origin. We hypothesized that its heterologous expression in *E. coli* led to poor solubility and misfolding, limiting the amount of active enzyme(Du et al., 2021). To solve this, we designed a new construct where a Thioredoxin A (TrxA) solubility-enhancing tag was fused to the N-terminus of *futC*(Figure 11).

![Cycle 1.4: Enhancing Key Enzyme Solubility with a Fusion Tag Diagram](https://static.igem.wiki/teams/5986/engineering/img13.webp)

Figure 11: Methods for the effect of TrxA tag fusion *futC* on 2 '-fucosyllactose yield. (A)Experimental Methods; (B)Experimental Flowchart; (C) The gene circuit of DH5α-*manB+manC*+*gmd*+*fcl*+TrxA-*futC*.

### Build

Using standard molecular cloning, we replaced the original *futC* gene in our plasmid with the newly designed *TrxA-futC* fusion gene. The resulting plasmid was transformed into our optimized *E. coli* DH5α chassis.

### Test

A comparative fermentation was conducted between the DH5α strain expressing the untagged *futC* and the new strain expressing the TrxA-*futC* fusion. Final 2'-FL yields were measured after 72 hours.

![Cycle 1.4: Enhancing Key Enzyme Solubility with a Fusion Tag Diagram](https://static.igem.wiki/teams/5986/engineering/img14.webp)

Figure 12: Comparison of 2'-FL production with and without the TrxA fusion tag.

The TrxA-*futC* strain yielded 279.47 ± 22.09 mg/L, a 56% increase over the untagged version (179.27 ± 11.58 mg/L)(Figure 12).

### Learn

The significant 56% increase in yield conclusively demonstrated that the solubility of the *futC* enzyme was indeed a major bottleneck. Our protein engineering strategy was successful. This final optimization completed our DBTL cycle for the 2'-FL module, resulting in a robust, high-yielding production strain and a clear, logical engineering workflow.

# System 2: Production of Functional Recombinant Trypsin

This system involved a complete "gene-to-function" engineering cycle to produce an active protease for improving protein digestion.

### Design

Our goal was to produce active trypsin. To avoid the toxicity of an active protease to the host cell, we designed a system to express its inactive precursor (Choi & Lee et al., 2004), trypsinogen (Part: BBa\_25FE8YUB, *TRYP*). The porcine *TRYP* gene was codon-optimized for *E. coli* and cloned into a pET-28a vector for IPTG-inducible expression in the BL21(DE3) host(Figure 13).

![System 2: Production of Functional Recombinant Trypsin Diagram](https://static.igem.wiki/teams/5986/engineering/img15.webp)

Figure 13: Construction of BL21-*TRYP*. (A) The plasmid map of pET28a-*TRYP*; (B) The gene circuit of BL21-*TRYP*; (C) The flow chart of Construction of BL21-*TRYP*; (D)the agarose gel electrophoresis analysis of the *TRYP* gene fragment

### Build

The pET-28a-*TRYP* vector was successfully constructed and transformed into *E. coli* BL21(DE3). High-level expression resulted in the formation of insoluble inclusion bodies. We therefore developed and optimized a multi-step downstream process(Figure 14):

Inclusion Body Isolation: Cells were lysed, and insoluble protein aggregates were collected by centrifugation.

Denaturation & Refolding: Inclusion bodies were solubilized in 8M urea and then refolded by rapid dilution into a redox buffer containing a cystine/cysteine pair to facilitate correct disulfide bond formation (Burgess et al., 2009).

Autocatalytic Activation: The refolded, inactive trypsinogen was transferred to an activation buffer where it autocatalytically cleaved its propeptide to become fully active trypsin.

![System 2: Production of Functional Recombinant Trypsin Diagram](https://static.igem.wiki/teams/5986/engineering/img16.webp)

Figure 14: This is a flow chart of "Trypsin Expression and Refolding".

### Test

We validated our system at both the protein and functional levels.

Protein Expression: Western Blot analysis confirmed the successful expression of the His-tagged *TRYP* protein at its expected molecular weight of ~26 kDa(Figure 15).

![System 2: Production of Functional Recombinant Trypsin Diagram](https://static.igem.wiki/teams/5986/engineering/img17.webp)

Figure 15: (A)The flow chart of Western Blot; (B)SDS-PAGE analysis of recombinant *TRYP* overexpression in E. coli

Enzymatic Activity: We performed a functional assay using the substrate N-Benzoyl-L-Arginine-Ethylester (BAEE). The absorbance change at 253 nm was measured to quantify activity(Figure 16). Enzymatic activity of the constructed engineered strain was determined to be 1748 U/mg.

![System 2: Production of Functional Recombinant Trypsin Diagram](https://static.igem.wiki/teams/5986/engineering/img18.webp)

Figure 16: (A)The flow chart of "Trypsin Activity Assay"; (B)Comparison of BAEE cleavage activity of intracellular proteins between BL21 (DE3) and *TRYP*-overexpressing strains

### Learn

The results provided a powerful demonstration of a complete gene-to-function workflow. We successfully expressed the target gene, established an effective protocol to recover active protein from inclusion bodies, and confirmed the high catalytic activity of the final product. This validated our design and provided a functional enzyme ready for its intended application in hydrolyzing casein.

# System 3: Functional Characterization of β-Galactosidase (*lacZ*)

This cycle focused on producing and validating the function of β-galactosidase to address lactose intolerance(Kumar et al., 2022).

### Design

Our objective was to produce active β-galactosidase (*lacZ*) using the existing BioBrick part BBa\_I732005 (Juers et al., 2012). We designed a system to express this part by cloning the *lacZ* gene into the pET-28a vector for high-level expression in *E. coli* BL21(DE3)(Figure 17).

![System 3: Functional Characterization of β-Galactosidase (<em>lacZ</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img19.webp)

Figure 17: Construction of BL21-*LacZ*. (A) The plasmid map of pET28a-*LacZ*; (B) The gene circuit of BL21-*LacZ*; (C)The flow chart of Construction of BL21-*LacZ*; (D) the agarose gel electrophoresis analysis of the *lacZ* gene fragment

### Build

The *lacZ* gene was successfully cloned into the pET-28a vector and transformed into *E. coli* BL21(DE3).

### Test

We validated our system at both the protein and functional levels.

TestProtein Expression: Western Blot analysis confirmed the expression of a protein at the expected size of β-galactosidase (~116 kDa). The expression of the full-length protein was verified (Figure 18).

![System 3: Functional Characterization of β-Galactosidase (<em>lacZ</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img20.webp)

Figure 18: (A) This is a flow chart of Western Blot; (B) SDS-PAGE analysis of recombinant *lacZ* overexpression in E. coli.

Functional Assay: We performed an in vitro functional assay to measure the enzyme's ability to hydrolyze lactose. The purified *lacZ* enzyme was incubated with a 10 mM lactose solution, and the lactose concentration was monitored over an 8-hour period(Figure 19).

![System 3: Functional Characterization of β-Galactosidase (<em>lacZ</em>) Diagram](https://static.igem.wiki/teams/5986/engineering/img21.webp)

Figure 19: (A) This is a flow chart of measurement of the lactose content *lacZ* breaks down; (B) Efficacy of recombinant β-galactosidase in lactose degradation

Efficacy of recombinant β-galactosidase in lactose degradation. The enzyme successfully degraded 80% of the initial lactose substrate (from 10 mM to ~2 mM) within 8 hours(Figure 19).

### Learn

The results clearly demonstrated that our engineered and purified *lacZ* protein is highly active and functional. The successful in vitro validation confirms that our recombinant enzyme is a viable candidate for its intended application: breaking down lactose in dairy products. This work also provides valuable new characterization data for the existing BioBrick part BBa\_I732005.

We plan to use Escherichia coli Nissle 1917 as the host strain to construct a stable and efficient engineered strain for large-scale production of 2′-FL, trypsin, and lactose. As a GRAS-certified probiotic, Nissle 1917 has good safety and metabolic engineering potential, making it ideal for food production. By optimizing key metabolic pathways, we aim to improve the synthesis efficiency and yield of 2′-FL, trypsin, and lactose, and facilitate the transition from laboratory-scale to industrial production. The goal is to achieve high-purity, cost-effective products that meet the nutritional needs of infants and promote gut health. This platform will also provide a scalable route for the synthesis of other human milk oligosaccharides (HMOs). Currently, we are using BL21 and DH5α strains for our experiments. Though the probiotic strain is safe for food use, like other Gram-negative bacteria, it may produce endotoxins. To ensure the safety of infant formulas, we will reduce endotoxin levels by modifying related genes and optimizing purification steps, establishing a safe and efficient system for the production of 2′-FL, trypsin, and lactose for nutrition and health applications.

# Reference

1. Bonin, C. P., Potter, I., Vanzin, G. F., & Reiter, W. D. (1997). The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4, 6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose. Proceedings of the National Academy of Sciences, 94(5), 2085-2090. [https://www.sci-hub.se/https://www.pnas.org/doi/abs/10.1073/pnas.94.5.2085](https://www.sci-hub.se/https%3A//www.pnas.org/doi/abs/10.1073/pnas.94.5.2085)
2. Burgess, R. R. (2009). Refolding solubilized inclusion body proteins. Methods in Enzymology, 463, 259–282.
3. Byun, S., Kim, M., Lee, W., Lee, K., Han, N. S., & Seo, J. (2006). Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli. Applied Microbiology and Biotechnology, 74(4), 768–775. <https://doi.org/10.1007/s00253-006-0730-x>
4. Chin, Y. W., Kim, J. Y., Lee, W. H., & Seo, J. H. (2015). Enhanced production of 2′-fucosyllactose in engineered Escherichia coli BL21star (DE3) by modulation of lactose metabolism and fucosyltransferase. Journal of Biotechnology, 210, 107-115. [https://www.sci-hub.se/https://www.sciencedirect.com/science/article/pii/S0168165615300547](https://www.sci-hub.se/https%3A//www.sciencedirect.com/science/article/pii/S0168165615300547)
5. Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins in Escherichia coli. Applied Microbiology and Biotechnology, 64(5), 625–635.
6. Du, F., Liu, Y. Q., Xu, Y. S., Li, Z. J., Wang, Y. Z., Zhang, Z. X., & Sun, X. M. (2021). Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production. Microbial cell factories, 20(1), 189. <https://link.springer.com/content/pdf/10.1186/s12934-021-01680-6.pdf>
7. Juers, D. H., Hakda, S., Matthews, B. W., & Huber, R. E. (2012). A structural view of the action of Escherichia coli (LacZ) β-galactosidase. Biochemistry, 51(31), 6086–6098.
8. Kumar, M., Tomar, S. K., & Singh, A. K. (2022). Lactose Hydrolysis in Dairy Products: A Review. In Enzymes in Food Biotechnology. Springer, Singapore.
9. Li, L., Kim, S. A., Fang, R., & Han, N. S. (2018). Expression of manB Gene from Escherichia coli in Lactococcus lactis and Characterization of Its Bifunctional Enzyme, Phosphomannomutase. Journal of Microbiology and Biotechnology, 28(8), 1293–1298. <https://doi.org/10.4014/jmb.1804.04030>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

Content

* [Genetic Engineering](#section-1)
  + [Bacterial Culture](#section-2)
    - [Bacterial Dry Weight Measurement](#section-3)
      * [Lactose Content Analysis](#section-4)
        + [HPLC Analysis of Fermentation Products](#section-5)
          - [2'-Fucosyllactose Content Assay](#section-6)
            * [Construction of the Trypsin-Expressing Engineered Bacterium](#section-7)
              + [Trypsin Expression and Refolding](#section-8)
                - [Trypsin Activity Assay](#section-9)
                  * [Construction of the LacZ-Expressing Engineered Strain](#section-10)
                    + [Western Blot Analysis of LacZ Expression](#section-11)
                      - [Measurement of Lactose Degradation by LacZ](#section-12)
                        * [Statistical Analysis](#section-13)

# Genetic Engineering

The genes *manB*, *manC*, *gmd*, *fcl*, and *futC* were synthesized by General Biosystems (China). The sequences were codon-optimized for *Escherichia coli* expression, and the restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were cloned into the pET28a vector via the NdeI and XhoI restriction sites. *E. coli* BL21 (DE3) (Cat. No. D1009S) and DH5α (Cat. No. D0351) supercompetent cells were purchased from Beyotime (China). Subsequently, the recombinant plasmids were transformed into *E. coli* DH5α and BL21 (DE3) cells. Positive clones were selected on Luria Bertani (LB) agar plates containing 100 μg/mL kanamycin (Kana) and were verified by sequencing (Tsingke, Beijing) to obtain the recombinant engineered strains. The strains were stored at -80°C with 25% (v/v) glycerol as a cryoprotectant. The engineered strains were cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium (Cat. No. G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin.

# Bacterial Culture

The recombinant strains were cultured in 50 mL Erlenmeyer flasks containing 10 mL of LB medium with kanamycin at 25°C and 250 rpm. The cell concentration was measured by the optical density (OD) at 600 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). When the cell OD600 reached 0.6, the bacteria were collected by centrifugation at 4000 × g for 5 min and resuspended in 100 mL of M9 Broth (supplemented with 1 mM MgSO4 and 50 μM CaCl2) (Cat. No. A510881, Sangon Biotech, China) containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 8 g/L lactose were added to induce the production of 2′-fucosyllactose (2-FL).

# Bacterial Dry Weight Measurement

The cell optical density (OD600) was measured at 600 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). Subsequently, the bacterial pellet was collected by centrifugation at 10,000 × g for 10 min and placed in an oven at 50°C for 8 h to measure the dry cell weight (DCW) of the recombinant *E. coli* strain.

# Lactose Content Analysis

The 2-FL engineered strain was inoculated into liquid LB medium containing 100 μg/mL kanamycin (Kana) and cultured at 25°C and 250 rpm. When the cell OD600 reached 0.6, the culture was transferred to 100 mL of M9 Broth containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM IPTG and 8 g/L lactose were added. 1 mL of the fermentation broth was collected at regular intervals. After centrifugation at 10,000 × g for 1 min, the culture supernatant was collected and stored at -20°C. The change in lactose content was analyzed using a lactose assay kit (Cat. No. JL-T1072, Jonlin). First, lactose is hydrolyzed by lactase to produce free galactose, which is further oxidized by galactose oxidase to form a product with an absorption peak at 570 nm.

Glucose-assay-protocol-book-v2b-ab272532 (website).pdf

0.1MB

[Download](https://static.igem.wiki/teams/5986/experiments/jl-t1072-lactose-48.pdf)

Principle of the Assay Kit: Lactose is decomposed into galactose and glucose under the action of β-galactosidase. Glucose is oxidized by a specific enzyme to produce a (pink) red product that reacts with a chromogenic agent. This product has a maximum absorption peak at 510 nm. The lactose content is then obtained by correcting for the background value of free glucose.

# HPLC Analysis of Fermentation Products

A High-Performance Liquid Chromatography (HPLC) system (WATERS) was used to analyze the 2'-fucosyllactose produced by the engineered strain. The system was equipped with a WATERS ACQUITY UPLC BEH AMIDE column (2.1 × 100 mm, 1.7 μm) and a refractive index detector (RID). 500 μL of the fermentation sample (broth) was mixed with 500 μL of acetonitrile. Then, after centrifugation at 12,000 × g for 5 minutes, the supernatant was filtered through a 0.22 μm filter membrane. The mobile phase was A: 80% acetonitrile and 20% ammonia water (0.1%); B: 30% acetonitrile and 70% ammonia water (0.1%). The samples were detected at a flow rate of 0.3 mL/min and a temperature of 45°C. According to the program gradient, from 0-7 min, mobile phase A decreased from 90% to 60%, and from 7-10 min, it was restored to 90%. From 0-7 min, mobile phase B increased from 10% to 40%, and from 7-10 min, it decreased to 10%.

|
|  |

Time (min) |

Buffer A（%） |

Buffer B（%） |

|
|  |

0 |

90 |

10 |

|
|  |

1 |

80 |

20 |

|
|  |

7 |

60 |

40 |

|
|  |

10 |

90 |

10 |

# 2'-Fucosyllactose Content Assay

The 2-FL engineered strain was inoculated into liquid LB medium containing 100 μg/mL kanamycin (Kana) and cultured at 25°C and 250 rpm. When the cell OD600 reached 0.6, the cells were centrifuged and resuspended in 100 mL of M9 Broth containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM IPTG and 8 g/L lactose were added. 1 mL of the fermentation broth was collected at regular intervals. 1 mL of the fermentation broth was collected. After centrifugation at 10,000 × g for 1 min, the culture supernatant was collected and stored at -20°C. The 2-FL content in the fermentation broth of the engineered strain was analyzed using a 2′-Fucosyllactose (2′-FL) Assay Kit (Cat. No. 680112, Synome) according to the manufacturer's instructions.

Glucose-assay-protocol-book-v2b-ab272532 (website).pdf

0.1MB

[Download](https://static.igem.wiki/teams/5986/experiments/2-fl-2-0.pdf)

# Construction of the Trypsin-Expressing Engineered Bacterium

The open reading frame (ORF) of TRYP (UniProt: P00761) was codon-optimized to comply with the RFC#10 standard, synthesized, and inserted into the NdeI and XhoI digested pET-28a plasmid to generate the pET-28a-TRYP recombinant vector. The vector was transformed into the *E. coli* BL21 (DE3) (Cat. No. D1009S, Beyotime, China) strain. Positive clones were selected on Luria Bertani (LB) agar plates containing 100 μg/mL kanamycin (Kana) and were verified by sequencing (Tsingke, Beijing) to obtain the recombinant engineered strain. The strain was stored at -80°C with 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium supplemented with 100 μg/mL kanamycin.

# Trypsin Expression and Refolding

Expression and Collection of Inclusion Bodies: BL21-pET-28a-TRYP was inoculated into a 250 mL flask containing 50 mL of LB medium with 100 μg/mL kanamycin and cultured in a shaker at 37°C and 150 rpm. When the OD600 reached 0.6, 0.5 mM IPTG was added, and the culture temperature was adjusted to 16°C for a 20 h induction. Subsequently, the bacterial pellet was collected by centrifugation (10,000 × g, 5 min) and resuspended in pre-chilled PBS (pH = 7.5). Sonication was performed using an ultrasonic cell disruptor (Jingxin, Shanghai, China) (70 W, 3 s interval, 1 s pulse, 20 min). Subsequently, the inclusion body pellet was collected by centrifugation at 10,000 × g for 30 min at 4°C.

Inclusion Body Refolding: The pellet was washed with PBS containing 2 M urea (Cat. No. A68476, Innochem, China) and incubated for 1 h at 25°C and 150 rpm. Subsequently, it was centrifuged at 10,000 × g for 30 min at 4°C. 10 mL of denaturation solution (20 mM Tris, 8 M urea, 20 mM DTT, pH 8.5) was added per gram of inclusion body pellet, and it was incubated for 4 hours at 25°C on a destaining shaker. The supernatant was collected after centrifugation at 12,000 rpm for 20 min. The supernatant was added to the refolding buffer (20 mM Tris, 1 mM cystine, and 3 mM cysteine, 1 M urea; pH = 9) at a 1:40 dilution ratio and stirred overnight at 4°C.

Zymogen Activation: TRYP was further concentrated using a 15 mL ultrafiltration tube (10 kDa, Millipore) and resuspended in Tris buffer (40 mM Tris, 0.1 M NaCl, 10 mM CaCl2, pH 8.0). After activation for 1 hour at 25°C, the protein concentration was determined using a Bradford assay kit (Cat. No. P0006, Beyotime, China). It was aliquoted and stored frozen at -80°C.

Explanation of Zymogen Activation Mechanism: In this experimental protocol, the activation of trypsinogen is based on a self-catalytic conformational transition mechanism: the trypsinogen, concentrated by ultrafiltration, is activated in a specific buffer system (40 mM Tris, 0.1 M NaCl, 10 mM CaCl₂, pH 8.0) at 25°C through the self-cleavage of the N-terminal propeptide sequence.

Specifically, Ca²⁺ in the buffer stabilizes the spatial conformation of trypsinogen, promoting the exposure of its catalytic active site; the weakly alkaline environment (pH 8.0) facilitates intermolecular interactions among trypsinogen molecules, initiating the spontaneous cleavage of the propeptide in a small number of molecules to generate active trypsin. The generated trypsin, through a positive feedback loop, further efficiently cleaves the propeptides of the remaining trypsinogen, ultimately completing the conversion from the inactive zymogen to the active trypsin. This process does not require an external activator (such as enterokinase) and achieves self-activation of the zymogen by optimizing the in vitro environment (ionic strength, temperature, pH).

What are inclusion bodies: Inclusion bodies are insoluble, inactive protein aggregates that form in the cytoplasm or periplasmic space when cells express recombinant proteins, due to overly rapid protein synthesis, incorrect folding, or the lack of necessary folding-assisting factors. They are typically granular in appearance and require denaturation and refolding treatments to restore protein activity.

# Trypsin Activity Assay

The trypsin activity of TRYP was determined using a Trypsin (TRY) Activity Assay Kit (Cat. No. AKPR005M, boxbio) according to the instructions. Trypsin is capable of catalyzing the hydrolysis of the ester bond in N-Benzoyl-L-Arginine-Ethylester (BAEE) to produce N-Benzoyl-L-Arginine (BA), and the product has a characteristic absorption peak at 253 nm. After incubating for 5 min, the absorbance at 253 nm was measured using a microplate reader (FlexStation 3, Molecular Devices, USA). One unit of enzyme activity is defined as the amount of enzyme that causes an increase in absorbance of 0.0005 at 253 nm per minute per mg of protein in a 1 mL reaction system at 25°C.

Official website link and product documentation for the kit:<https://www.boxbio.cn/product/772.html?goodsno=AKPR005M>

boxbio-akpr005m-try.pdf

0.3MB

[Download](https://static.igem.wiki/teams/5986/experiments/boxbio-akpr005m-try.pdf)

Western Blot Analysis of TRYP Expression

The engineered bacterial strain overexpressing TRYP was inoculated into 50 mL of Luria-Bertani (LB) medium supplemented with ampicillin and cultured overnight. The following day, the bacterial culture was collected by centrifugation at 10,000 × g for 1 minute. The resulting cell pellet was resuspended in PBS and subsequently lysed by sonication (150 W, with cycles of 1 second on and 3 seconds off, for a total of 20 minutes). The lysed cell suspension was then transferred to a new centrifuge tube and designated as the whole-cell lysate sample.

The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 ratio and denatured in a boiling water bath for 15 minutes. Proteins were then separated by 12% SDS-PAGE at 120 V. Subsequently, the proteins were transferred from the SDS-PAGE gel to a polyvinylidene fluoride (PVDF) membrane under ice-cold conditions. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature.

Given that the pET vector incorporates a His-tag, the membrane was incubated overnight at 4°C with a mouse anti-His-tag primary antibody (1:1000 dilution, Cat# AH367, Beyotime). After incubation, the membrane was washed three times with TBST for 10 minutes each. It was then incubated for 1 hour at room temperature with a Horseradish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat# A0216, Beyotime).

Following three final 10-minute washes with TBST, the chemiluminescence signal was developed. ECL solutions A and B were mixed at a 1:1 ratio immediately before use. The washed PVDF membrane was briefly placed on absorbent paper to remove excess surface liquid, then laid on the tray of the imaging system. The prepared ECL substrate was applied to completely cover the membrane. After a 1-minute reaction, excess substrate was wicked away, and the membrane was placed into the chemiluminescence imaging system to capture the signal according to a preset program.

# Construction of the LacZ-Expressing Engineered Strain

The open reading frame (ORF) of *LacZ* was codon-optimized to conform to the RFC#10 standard, synthesized, and subsequently inserted into a pET-28a plasmid that had been digested with *Nde*I and *Xho*I, thereby generating the recombinant vector pET-28a-LacZ. This vector was then transformed into the *E. coli* BL21(DE3) strain (Cat# D1009S, Beyotime, China). Positive clones were selected on Luria-Bertani (LB) plates supplemented with 100 μg/mL kanamycin (Kan) and validated by sequencing (Tsingke, Beijing) to obtain the final recombinant engineered strain. The strain was preserved at -80°C using 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium containing 100 μg/mL Kan.

# Western Blot Analysis of LacZ Expression

The engineered bacterial strain overexpressing LacZ was inoculated into 50 mL of LB medium supplemented with ampicillin and cultured overnight. The following day, the bacterial culture was harvested by centrifugation at 10,000 × g for 1 minute. The cell pellet was resuspended in PBS and lysed by sonication (150 W, operating in cycles of 1 second on and 3 seconds off, for a total duration of 20 minutes). The resulting lysate was transferred to a new centrifuge tube to serve as the whole-cell lysate sample.

The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 (v/v) ratio and denatured in a boiling water bath for 15 minutes. The proteins were separated by 12% SDS-PAGE at 120 V. Subsequently, the proteins were transferred from the SDS-PAGE gel to a polyvinylidene fluoride (PVDF) membrane under ice-cold conditions. The membrane was blocked for 1 hour at room temperature with 5% non-fat milk prepared in TBST. As the pET vector contains a His-tag, the membrane was incubated overnight at 4°C with a mouse anti-His-tag primary antibody (1:1000 dilution, Cat# AH367, Beyotime). The membrane was then washed three times with TBST, for 10 minutes each. Following this, it was incubated for 1 hour at room temperature with a Horseradish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat# A0216, Beyotime).

After three final 10-minute washes with TBST, ECL solutions A and B were mixed at a 1:1 ratio for immediate use. The washed PVDF membrane was placed on absorbent paper to briefly remove excess surface liquid, then positioned on the tray of a chemiluminescence imaging system. The prepared ECL substrate was applied to ensure the membrane was completely submerged. After a 1-minute reaction, excess liquid was wicked away from the top, and the membrane was placed into the imaging system to initiate the chemiluminescence reaction according to a preset program.

# Measurement of Lactose Degradation by LacZ

Purified LacZ enzyme was added to a 1 mL reaction system containing an initial lactose concentration of 10 mM, achieving a final enzyme concentration of 0.1 U/mL. The reaction was incubated at an optimal temperature, and samples were collected at 0, 2, 4, 6, and 8 hours. The production of glucose, which serves as an indirect measure of lactose consumption, was quantified using the Abcam Glucose Assay Kit (Cat# ab272532).

The specific procedure was as follows: 5 μL of either the glucose standard or the test sample was added to a 1.5 mL centrifuge tube. To this, 500 μL of the detection reagent was added, and the solution was mixed by vortexing. The tube was then tightly capped and precisely heated in a boiling water bath for 8 minutes, followed by cooling in an ice-water bath for 4 minutes. A 200 μL aliquot of the reaction solution was transferred to a 96-well plate. The absorbance was measured at a wavelength of 630 nm using a microplate reader. The glucose concentration was calculated based on the standard curve, which was then used to determine the amount of lactose degraded.

Glucose-assay-protocol-book-v2b-ab272532 (website).pdf

0.1MB

[Download](https://static.igem.wiki/teams/5986/experiments/glucose-assay-protocol-book-v2b-ab272532-website.pdf)

# Statistical Analysis

GraphPad Prism software was used for data analysis and plotting. Data are presented as mean ± standard deviation (SD). For comparisons of multiple groups, one-way analysis of variance (One-way ANOVA) followed by Tukey's post hoc test was used for difference analysis. For comparisons between two groups, a Student's t-test was used for analysis. A p-value of less than 0.05 was considered statistically significant.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

Content

* [Abstract](#section-1)
  + [System 1: 2'-FL production](#section-2)
    - [Comparison of 2'-fucosyllactose(2'-FL) yield in BL21-*manB*+*manC* after 72-hour fermentation](#section-3)
      * [Objective](#subsection-4)
      * [Methods](#subsection-5)
      * [Results](#subsection-6)
      * [Conclusion](#subsection-7)
    - [Yield comparison of 2 '-fucosyllactose after 72 hours of fermentation of BL21 recombinant strain](#section-8)
      * [Objective](#subsection-9)
      * [Methods](#subsection-10)
      * [Results](#subsection-11)
      * [Conclusion](#subsection-12)
    - [Changes in 2'-FL yield, Lactose Content, and Cell Dry Weight Over Time in BL21-*manB+man**C*+*gmd*+*fcl*+*futC*](#section-13)
      * [Objective](#subsection-14)
      * [Methods](#subsection-15)
      * [Results](#subsection-16)
      * [Conclusion](#subsection-17)
    - [HPLC analysis of fermentation products](#section-18)
      * [Objective](#subsection-19)
      * [Methods](#subsection-20)
      * [Results & Conclusion](#subsection-21)
    - [The effect of two different strains(BL21/DH5α) on 2'FL yield](#section-22)
      * [Objective](#subsection-23)
      * [Methods](#subsection-24)
      * [Results](#subsection-25)
      * [Conclusion](#subsection-26)
    - [The effect of TrxA tag fusion *futC* on 2 '-fucosyllactose yield](#section-27)
      * [Objective](#subsection-28)
      * [Methods](#subsection-29)
      * [Results](#subsection-30)
      * [Conclusion](#subsection-31)
    - [Summary](#section-32)
      * [System 2: Trypsin](#section-33)
        + [Construction of trypsin Expression Vector](#subsection-34)
        + [Objective](#subsection-35)
        + [Methods](#subsection-36)
        + [Results](#subsection-37)
        + [Conclusion](#subsection-38)
        + [Objective](#subsection-39)
        + [Methods](#subsection-40)
        + [Results](#subsection-41)
        + [Conclusion](#subsection-42)
      * [The trypsin expression and refolding](#section-43)
        + [Objective](#subsection-44)
        + [Methods](#subsection-45)
        + [Results](#subsection-46)
        + [Conclusion](#subsection-47)
      * [Summary](#section-48)
        + [System 3: *lacZ*](#section-49)
          - [Construction of *lacZ* Expression Vector](#subsection-50)
          - [Objective](#subsection-51)
          - [Methods](#subsection-52)
          - [Result](#subsection-53)
        + [Conclusion](#section-54)
          - [Verification of Recombinant *lacZ* Protein Expression](#subsection-55)
          - [Objective](#subsection-56)
          - [Methods](#subsection-57)
          - [Western Blot](#subsection-58)
          - [Result](#subsection-59)
        + [Conclusion](#section-60)
          - [Functional Assay of Purified Recombinant *lacZ*](#subsection-61)
          - [Objective](#subsection-62)
          - [Methods](#subsection-63)
          - [Measurement of the lactose content *lacZ* breaks down](#subsection-64)
          - [Result](#subsection-65)
        + [Conclusion](#section-66)
          - [Summary](#section-67)
            * [Reference](#section-68)

# Abstract

This section presents the successful construction and functional validation of the three core modules designed for our "ProBabyotics" platform, aimed at systemically enhancing the nutritional value of infant formula. Each module was systematically engineered and rigorously tested, providing a robust proof-of-concept for our overall project.

For the Immune Enhancement Module (2'-FL): We successfully established a de novo biosynthetic pathway for 2'-fucosyllactose (2'-FL). Through a multi-stage DBTL (Design-Build-Test-Learn) engineering cycle, we progressively optimized the system. Key improvements included selecting a *lacZ*-deficient DH5α chassis to eliminate substrate competition (achieving a ~19-fold yield increase) and employing a TrxA fusion tag to enhance the solubility of the rate-limiting *futC* enzyme (a further 56% boost). This iterative process increased the 2'-FL titer from an initial 8.10 mg/L to a final yield of 279.47 ± 22.09 mg/L. The identity of the product was unequivocally confirmed by HPLC.

For the Protein Hydrolysis Module (Trypsin): We developed a complete "gene-to-function" pipeline for producing active recombinant trypsin. The codon-optimized *TRYP* gene was successfully cloned and its expression was confirmed via Western Blot. We established an effective protocol to refold the protein from inclusion bodies and activate it via autocatalysis. The resulting enzyme demonstrated high functionality, with a specific activity of 1748 U/mg in a BAEE hydrolysis assay.

For the Lactose Tolerance Module (β-Galactosidase): We successfully expressed and characterized β-galactosidase (*lacZ*) from the existing BioBrick part BBa\_I732005. After confirming the expression of the full-length, ~116 kDa protein via Western Blot, we performed an *in vitro* functional assay. The purified enzyme proved to be highly effective, degrading 80% of the lactose substrate within 8 hours.

Collectively, these results validate the feasibility of our modular approach. We have successfully produced and characterized all three key functional components, laying a solid scientific foundation for the development of our next-generation infant formula, "2'F-Luxe".

![Abstract  Diagram](https://static.igem.wiki/teams/5986/results/img1.webp)

Figure 1: ProBabyotics: Engineering a Better Formula

# System 1: 2'-FL production

This section details the systematic engineering of an *E. coli* strain for the de novo biosynthesis of 2'-fucosyllactose (2'-FL), a key human milk oligosaccharide(Yang et al., 2025). Our project followed a multi-round 'Design-Build-Test-Learn' (DBTL) cycle to progressively identify and overcome metabolic bottlenecks(Zhao et al., 2025)a. Key strategies included constructing a complete five-gene metabolic pathway, optimizing the host chassis to eliminate substrate competition, and employing protein engineering to enhance the activity of a rate-limiting enzyme. Through these iterative improvements, we successfully increased the 2'-FL titer from an initial proof-of-concept yield of 8.10 mg/L to a final yield of 279.47 ± 22.09 mg/L, demonstrating a clear and successful engineering workflow.

# Comparison of 2'-fucosyllactose(2'-FL) yield in BL21-*manB*+*manC* after 72-hour fermentation

## Objective

The objective of this experiment was to provide a proof-of-concept for our 2'-FL biosynthetic pathway. Specifically, we aimed to determine if the co-expression of the first two key enzymes, *manB* and *manC*, is sufficient to initiate the de novo synthesis of 2'-FL in our *E. coli* BL21 chassis.

## Methods

### -Genetic Engineering

Synthesize *manB* and *manC* and clone the pET28a vector through NdeI and XhoI restriction sites. *E. coli* BL21 (DE3) (D1009S) and DH5α (DE3) (D0351) supercompetent cells were purchased from Beyotime. Subsequently, the recombinant plasmid is transformed into *E. coli* DH5α and BL21. Positive clones are screened on LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), followed by sequencing verification (Beijing, Jingke) to obtain the recombinant engineering strain. The strain is stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.

![Methods  Diagram](https://static.igem.wiki/teams/5986/results/img2.webp)

Figure 2: Construction of BL21-*manB+manC*. (A) The plasmid map of pET28a-*manB+manC*; (B) The gene circuit of BL21-*manB+manC*; (C) Flow chart of Genetic engineering

### -Bacterial Culture

Transfer the recombinant strains in 50mL Erlenmeyer flasks containing 10 mL LB medium with Kana, under conditions of 25°C, 250 rpm. Measure cell concentration by optical density at 600 nm using a microplate reader （FlexStation 3，Molecular Devices，USA）. When OD₆₀₀ reaches 0.6, bacteria are collected by centrifugation at 4000 g for 5 min and resuspended into 100 mL M9 Broth (1 mM MgSO₄ and 50 μM CaCl₂)(A510881, Sangon Biotech, China) with 20 g/L glycerol and 5 g/L yeast extract. As cell OD₆₀₀ reached 0.6 again , 0.5 mM IPTG and 8 g/L lactose are added to produce 2'-fucosyllactose (2'-FL).

![Methods  Diagram](https://static.igem.wiki/teams/5986/results/img3.webp)

Figure 3: Flow chart of bacterial culture.

### -2'-FL content assay

To quantify the 2'-FL concentration, 1 mL of fermented broth was collected at designated intervals. The samples were centrifuged at 10,000 g for 1 minute, and the resulting supernatant was collected and stored at -20°C for subsequent analysis.

The 2'-FL content was determined using a commercial detection kit (Synome, 680112), which relies on a two-step enzymatic cascade. A highly specific α-L-fucosidase first hydrolyzes 2'-FL to release L-fucose. Subsequently, L-fucose dehydrogenase (FDH) oxidizes the L-fucose to L-fucono-1,5-lactone. This reaction is coupled with the stoichiometric reduction of NADP⁺ to NADPH. Since NADPH has a strong characteristic absorbance at 340 nm, the increase in absorbance is directly proportional to the amount of NADPH produced, which is equimolar to the initial amount of 2'-FL in the sample. The final concentration was calculated based on the change in absorbance at 340 nm (A₃₄₀).

![Methods  Diagram](https://static.igem.wiki/teams/5986/results/img4.webp)

Figure 4: Flow chart of 2'-FL content assay

## Results

The results of the agarose gel electrophoresis are shown in Figure 5. A distinct band of the expected size (1947 bp) was observed, confirming the successful express of *manB+manC*.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img5.webp)

Figure 5: the agarose gel electrophoresis analysis of the *manB+manC* gene fragment

The 2'-fucosyllactose content in both BL21 and BL21-p28a is close to 0 mg/L, while the average 2'-fucosyllactose content of BL21- *manB+manC* is 8.10±1.03 mg/L. 2'-fucosyllactose content is higher in BL21-*manB+manC* than in BL21 and BL21-p28a.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img6.webp)

Figure 6: Comparison of 2'-FL yield in BL21-*manB+manC* after 72-hour fermentation

## Conclusion

The results demonstrate that the co-expression of *manB* and *manC* is both necessary and sufficient to initiate 2'-FL synthesis in *E. coli* BL21. The engineered strain produced a measurable yield (8.10±1.03 mg/L), whereas the control strains showed negligible production. While modest, this yield serves as a crucial validation that the foundational step of our pathway is functional. This outcome strongly suggests that the downstream enzymes are the next rate-limiting step, guiding our subsequent work to incorporate the complete gene cassette (*gmd*, *fcl*, and *futC*) to improve the final yield(Moriyama et al., 2025).

# Yield comparison of 2 '-fucosyllactose after 72 hours of fermentation of BL21 recombinant strain

## Objective

This experiment aims to evaluate the effect of expression of different genes in BL21 recombinant strains on the yield of 2'-FL after 72 hours of fermentation.

## Methods

### -Genetic Engineering

Synthesize *manB, manC,* *gmd*，*fcl* and *futC,* and Clone the pET28a vector through NdeI and XhoI restriction sites. *E. coli* BL21 (DE3) (D1009S) and DH5α (DE3) (D0351) supercompetent cells were purchased from Beyotime. Subsequently, the recombinant plasmid is transformed into *E. coli* DH5α and BL21. Positive clones are screened on LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), followed by sequencing verification (Beijing, Jingke) to obtain the recombinant engineering strain. The strain is stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img7.webp)

Figure 7: Construction of BL21-*manB+manC*+*gmd*+*fcl*+*futC*. (A) The plasmid map of pET28a-*manB+manC*+*gmd*+*fcl*+*futC* (B) The gene circuit of BL21-*manB+manC*+*gmd*+*fcl*+*futC*.

### -Bacterial Culture

Transfer the recombinant strains in 50mL Erlenmeyer flasks containing 10 mL LB medium with Kana, under conditions of 25°C, 250 rpm. Measure cell concentration by optical density at 600 nm using a microplate reader （FlexStation 3，Molecular Devices，USA）. When OD₆₀₀ reaches 0.6, bacteria are collected by centrifugation at 4000 g for 5 min and resuspended into 100 mL M9 Broth (1 mM MgSO₄ and 50 μM CaCl₂)(A510881, Sangon Biotech, China) with 20 g/L glycerol and 5 g/L yeast extract. As cell OD₆₀₀ reached 0.6 again , 0.5 mM IPTG and 8 g/L lactose are added to produce 2'-fucosyllactose (2'-FL).

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img8.webp)

Figure 8: Flow chart of bacterial culture.

### -2'-FL content assay

Collect 1mL fermented broth from cultured bacteria at intervals. After centrifugation at 10,000g for 1 min, the supernatant is stored at -20°C. Calculate the 2'-FL content according to the instruction(680112, Synome).

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img9.webp)

Figure 9: Flow chart of 2'-FL content assay

## Results

The agarose gel electrophoresis results for the *manA*, *gmd*, *fcl* and *futC* gene are shown in Figure 10. As indicated by the marker, bands of the correct corresponding lengths were observed for all genes, confirming their successful expression.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img10.webp)

Figure 10: Agarose gel electrophoresis analysis of the *manA*, *gmd*, *fcl*, *futC* gene fragment

We synthesise *manA*, *manB, manC,* *gmd*，*fcl* and *futC* at first, but due to the significantly slow growth rate of the recombinant strain after expressing *manA**,* a literature review was conducted. It was determined that the *manA*gene is not essential for the 2'-FL production pathway. Therefore, the recombinant strain used for subsequent 2'-FL yield measurements contained the gene sequence *manB*, *manC* , *gmd*, *fcl*, and *futC.*

As shown in Figure 11, the results indicate that the 2'-FL content in both BL21 and BL21-p28a is close to 0 mg/L. And the average 2'-FL content of BL21-*manB+manC* and BL21-*manB+manC*+*gmd*+*fcl*+*futC* are 8.11±1.04 mg/L and 19.66±2.62 mg/L respectively. Overall, BL21-*manB+manC*+gmd+fcl+futC yields the most 2'-fucosyllactose.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img11.webp)

Figure 11: Yield comparison of 2 '-fucosyllactose after 72 hours of fermentation of BL21 recombinant strain

## Conclusion

The maximum yield of the recombinant strain BL21-*manB+manC*+gmd+fcl+futC reaches 19.66±2.62 mg/L of 2-FL. This indicates that overexpression of manB, manC, gmd, fcl and futC can significantly increase the yield of 2 '-FL, which is consistent with other findings(Liang et al., 2025).

# Changes in 2'-FL yield, Lactose Content, and Cell Dry Weight Over Time in BL21-*manB+man**C*+*gmd*+*fcl*+*futC*

## Objective

Building upon our initial proof-of-concept, this experiment aimed to quantify the impact of expressing the complete de novo synthesis pathway on 2'-FL production. We hypothesized that by co-expressing the downstream enzymes (*gmd*, *fcl* and *futC* ) alongside *manB* and *manC*, we could overcome the metabolic bottleneck identified previously and significantly increase the final 2'-FL yield.

## Methods

### -Engineered strain used

The BL21-*manB+manC*+*gmd*+*fcl*+*futC* engineered strain we used is manufactured in the previous steps.

### -Bacteria dry cell weight determination

Measure cell optical density at 600nm using a microplate reader （FlexStation 3，Molecular Devices，USA). During fermentation, 1 mL broth samples are collected at designated time intervals to monitor cell growth. The bacterial cells are immediately pelleted by centrifugation at 10,000 × g for 10 minutes. After carefully removing the supernatant, the cell pellets undergo a drying process in a 50°C oven for 8 hours to completely evaporate residual moisture. The resulting dried biomass is then precisely weighed to determine the dry cell weight (DCW), as

DCW(g/L)=(*W*tube+cell​−*W*tube) /*V*sample×dilution factor .

### ​-2'-FL content assay

Collect 1mL fermented broth from cultured bacteria at intervals. After centrifugation at 10,000g for 1 min, the supernatant is stored at -20°C. Then calculate the 2'-FL content according to the instruction(680112, Synome).

### -Lactose content assay

Repeat the first and second steps in the process '2'-FL content assay', follow by the calculation of lactose content according to the instruction(Jonln, JL-T1072).

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img12.webp)

Figure 12: Flow charts of bacteria dry cell weight determination, 2'-FL content assay and lactose content assay.

## Results

It is clear from the graph that during the period of 72 hours, the amount of lactose fall consistently while dry cell mass and the figure for 2’-FL and the dry cell mass rised from 0.00 mg/L to about 22.71 mg/L and from 0.00 mg/L to 18.00 mg/L respectively.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img13.webp)

Figure 13: Changes in 2'-FL yield, Lactose Content, and Cell Dry Weight Over Time in BL21-*manBC*+*gmd*+*fcl*+*futC*

## Conclusion

The results strongly support our hypothesis. The introduction of the complete five-gene cassette (*manB+manC*+*gmd*+*fcl*+*futC*) led to a final 2'-FL yield of 19.66±2.62 mg/L, a statistically significant (p < 0.0001) and 2.4-fold increase over the strain expressing only *manB+manC*. This demonstrates that the downstream enzymes, particularly the final fucosyltransferase (*futC*), are crucial for converting the metabolic intermediates into the final product.

Additionally, we learned that including *manA* in the construct severely impaired cell growth, and a literature review confirmed it was non-essential (Li et al., 2025). This finding refined our final construct design. Overall, we successfully established a functional, multi-gene pathway for 2'-FL synthesis in *E. coli*, laying the groundwork for further optimization.

# HPLC analysis of fermentation products

## Objective

While the enzymatic assay provided a convenient method for high-throughput quantification, its detection principle is based on the measurement of L-fucose released after hydrolysis. Consequently, the assay cannot distinguish authentic 2'-FL from other potential fucose-containing byproducts (such as other fucosylated oligosaccharides) or any pre-existing free L-fucose in the fermentation broth. This inherent lack of specificity creates potential for interference and necessitates a more rigorous confirmation of product identity.

Therefore, to provide definitive and orthogonal validation, we employed High-Performance Liquid Chromatography (HPLC). The objective of this experiment was to separate the components of our fermentation supernatant and compare the retention time of the major product peak against that of a pure 2'-FL analytical standard. This method provides unambiguous chemical identification, confirming that the compound we quantified was indeed our target molecule, 2'-FL. Given the complexity of the instrumentation and method development, these experiments were conducted with the invaluable guidance of our instructors.

![Objective Diagram](https://static.igem.wiki/teams/5986/results/img14.webp)

Figure 14: Students performed the complex HPLC analysis under the guidance of our instructors.

## Methods

The recombinant strains mentioned above are supplemented into the LB medium containing Kana, under the conditions of 25°C and 250 rpm. Detect the value of OD₆₀₀ and as it reaches a0.6, transfer the strains to M9 medium (20 g/L glycerol, 5 g/L yeast extract, 1 mM MgSO4, 50 μM CaCl2). As OD₆₀₀=0.6 again, the production of 2’-FL will be induced by adding 0.5 mM IPTG and 8 g/L lactose.The production of 2'-FL by engineered bacteria will be analyzed by a high performance liquid chromatography (HPLC) system (WATERS), equipped with a WATERS ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm). The mobile phases are A: 80% acetonitrile and 20% ammonia (0.1%), B: 30% acetonitrile and 70% ammonia (0.1%), with flow rate of 0.3 mL/min and temperature of 45°C. According to the programmed gradient, mobile phase A decreases from 90% to 60% at 0-7 min and recoveres to 90% at 7-10 min. Mobile phase B increases from 10% to 40% at 0-7 min and decreases to 10% at 7-10 min.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img15.webp)

Figure 15: Mechanism of HPLC

## Results & Conclusion

The HPLC chromatogram of the fermentation supernatant from our engineered BL21 strain is presented in Figure 16. A prominent peak was detected at a retention time of 4.25 minutes. This retention time perfectly matched that of the 2'-FL analytical standard run under identical conditions.

This result provides definitive evidence that our engineered metabolic pathway is functional and that the compound being produced is, in fact, 2'-FL. The successful verification of the product's identity is a critical milestone, validating our quantitative yield measurements and confirming the success of our genetic design.

![Results & Conclusion Diagram](https://static.igem.wiki/teams/5986/results/img16.webp)

Figure 16: HPLC analysis of fermentation products of BL21

# The effect of two different strains(BL21/DH5α) on 2'FL yield

## Objective

Based on the knowledge that the *lacZ* gene in *E. coli* BL21 creates a competitive pathway for our lactose substrate, we hypothesized that switching to a *lacZ* -deficient chassis would significantly increase 2'-FL yield. This experiment was designed to directly test this hypothesis by comparing the productivity of our engineered pathway in *E. coli* BL21 versus *E. coli* DH5α under identical fermentation conditions(Chen et al., 2024).

## Methods

Our group used the same method to engineer 2 kinds of bacteria: BL21-*manB+manC*+*gmd*+*fcl*+*futC* and *DH5*α*-manB+manC*+*gmd*+*fcl*+*futC* by transferring designed recombinant plasmid to competentE.coli BL21 and DH5α. The concentration of 2'-FL in the sample is detected through absorbance , using a reagent kit and a microplate reader.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img17.webp)

Figure 17: Methods for Exploring the effect of two different strains(BL21/DH5α) on 2'FL yield. (A)Experimental Methods; (B)Experimental Flowchart

## Results

We conducted a head-to-head comparison by fermenting our engineered BL21 and DH5α strains under identical conditions. The results were definitive: the DH5α strain produced 158.26 ± 21.67 mg/L of 2'-FL, a nearly 19-fold improvement over the 8.23 ± 1.86 mg/L from the BL21 strain (Figure 18).

![Results Diagram](https://static.igem.wiki/teams/5986/results/img18.webp)

Figure 18: The effect of two different strains(BL21/DH5α) on 2'FL production

## Conclusion

The results provide a definitive validation of our hypothesis. The *lacZ*-deficient DH5α strain produced 158.26 ± 21.67 mg/L of 2'-FL, a remarkable ~19-fold increase compared to the BL21 strain. This demonstrates that chassis selection is a critical design parameter in metabolic engineering. By simply eliminating a single competing enzyme (*lacZ*), we successfully redirected the metabolic flux towards our desired product, achieving a substantial improvement in overall pathway efficiency. This learning was immediately incorporated into our subsequent engineering cycles.

# The effect of TrxA tag fusion *futC* on 2 '-fucosyllactose yield

## Objective

The *futC* gene, which encodes the final and rate-limiting enzyme in our pathway, is of human origin. Heterologous expression of such proteins in *E. coli* often leads to misfolding and poor solubility. To address this anticipated bottleneck, we employed a protein engineering strategy (Li et al., 2024). This experiment aimed to test the hypothesis that fusing a Thioredoxin A (TrxA) solubility-enhancing tag to the N-terminus of *futC* would improve its functional expression and thereby boost the final 2'-FL yield in our optimized DH5α chassis.

## Methods

First, we used the same method described above to construct the engineered strain DH5α and overexpress the *manB+manC*+*gmd*+*fcl*+*futC* gene. Then, we introduced *TrxA* tag fusion into DH5α. We detect the production of 2'-FL by using the 2'-FL Detection Kit according to the manufacturer’s instructions.

![Methods  Diagram](https://static.igem.wiki/teams/5986/results/img19.webp)

Figure 19: Methods for the effect of TrxA tag fusion *futC* on 2 '-fucosyllactose yield. (A)Experimental Methods; (B)Experimental Flowchart; (C) The gene circuit of DH5α-*manB+manC*+*gmd*+*fcl*+TrxA-*futC*.

## Results

The results showed that fusing *futC* with a TrxA tag significantly boosted 2'-FL production. The engineered strain with the TrxA-*futC* fusion yielded 279.47 ± 22.09 mg/L, a 56% improvement over the control strain expressing untagged *futC* (179.27 ± 11.58 mg/L), as detailed in Figure 20.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img20.webp)

Figure 20: The effect of TrxA tag fusion *futC* on 2 '-fucosyllactose yield

## Conclusion

The data conclusively demonstrates the success of our protein engineering approach. The strain expressing the TrxA-futC fusion protein yielded 279.47 ± 22.09 mg/L of 2'-FL, achieving a significant 56% increase over the control with untagged *futC*.

This result confirms that the poor solubility of the heterologously expressed *futC* was indeed a major limiting factor. By leveraging the TrxA fusion tag, we successfully increased the amount of soluble, active *futC* enzyme in the cell. This serves as a powerful demonstration of how targeted protein engineering can be used to overcome expression challenges and unlock the full potential of a metabolic pathway (Young & Robinson, 2014).

# Summary

In summary, our team successfully executed a four-stage engineering cycle that progressively improved 2'-FL production by over 34-fold. Each stage provided critical insights that guided the next round of design:

Pathway Completion: We first established that the full five-gene cassette was essential, increasing the yield by 2.4-fold compared to the initial two-gene construct. This confirmed that downstream enzymes were a primary bottleneck.

Chassis Optimization: We demonstrated the critical impact of host chassis selection. Switching to the *lacZ*-deficient DH5α strain eliminated substrate competition for lactose, resulting in a remarkable ~19-fold yield increase and identifying a major point of metabolic inefficiency (Chen et al., 2024).

Protein Engineering: We confirmed that the heterologous expression of the human *futC* enzyme was another limiting factor. By fusing it with a TrxA solubility tag, we successfully improved its functional expression, boosting production by a further 56%(Li et al., 2024).

Collectively, these results provide a robust, step-by-step validation of our metabolic engineering strategy. We have not only created a high-performing production strain but also documented a clear and logical workflow that can serve as a valuable reference for future iGEM teams tackling complex pathway optimization challenges (Gao et al., 2025).

# System 2: Trypsin

This section details the complete workflow for the production of a functional recombinant protease, trypsin, designed to improve protein digestion in infant formula (Jakobsson et al., 2000). Our engineering process encompasses the entire journey from gene to function: (1) plasmid construction of a codon-optimized porcine trypsinogen (*TRYP*) gene; (2) protein expression verification in *E. coli* BL21(DE3) via Western Blot; and (3) a multi-step process of protein purification, refolding, and activation from inclusion bodies. The success of this workflow was ultimately validated by a quantitative enzymatic assay, which confirmed the high catalytic activity of our final product (Rawlings et al., 1994).

## Construction of trypsin Expression Vector

## Objective

This experiment aimed to construct a recombinant plasmid for the expression of porcine trypsinogen (*TRYP*) and verify the successful cloning of the *TRYP* gene. The goal was to create a foundational tool, the pET-28a-*TRYP* vector, which would serve as the basis for producing recombinant trypsin in our *E. coli* BL21(DE3) chassis.

## Methods

### -The construction of trysin-engineered bacteria

The *TRYP* gene (Uniprot: P00761) is codon-optimized according to RFC10 standards, synthesized, and cloned into the NdeI/XhoI-digested pET-28a plasmid to construct the pET-28a-*TRYP* recombinant vector (Buck et al., 1962). This vector is then transformed into E. coli BL21(DE3) competent cells (Beyotime, D1009S). Positive transformants are selected on LB agar plates supplemented with 100 μg/mL Kana and subsequently verified by sequencing (Qingke, Beijing).

The confirmed recombinant strain is preserved at -80°C in 25% (v/v) glycerol for long-term storage. For routine cultivation, the engineered strain is grown in LB liquid medium containing 100 μg/mL Kana at 37°C with shaking at 150 rpm.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img21.webp)

Figure 21: Construction of BL21-*TRYP*. (A) The plasmid map of pET28a-*TRYP*; (B) The gene circuit of BL21-*TRYP*; (C) The flow chart of Construction of BL21-*TRYP*.

## Results

The agarose gel electrophoresis results for the *TRYP* gene are shown in Figure 22. As indicated by the marker, bands of the correct corresponding lengths were observed for *TRYP* genes, confirming it's successful expression.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img22.webp)

Figure 22: the agarose gel electrophoresis analysis of the *TRYP* gene fragment

## Conclusion

The agarose gel electrophoresis result confirms the successful construction of the pET-28a-*TRYP* expression vector. A single, bright band was observed at the expected size of 693 bp, which corresponds to the length of the codon-optimized *TRYP* gene. This result validates that the *TRYP* gene was successfully amplified and cloned into the pET-28a plasmid using standard methods (Sambrook & Russell, 2001), enabling us to proceed with transforming the vector into our expression host for protein production and functional analysis.

### Verification of Recombinant *TRYP* Protein Expression

## Objective

Following the successful construction of the pET-28a-*TRYP* vector, this experiment aimed to verify the expression of the recombinant, His-tagged *TRYP* protein at the protein level. Western Blot analysis was employed to specifically detect the target protein in the cell lysate of our engineered *E. coli* strain, confirming that the genetic construct is functional and produces the intended protein(Nyaruhucha et al., 1997)..

## Methods

The *TRYP*-overexpressing engineered strain was inoculated into 50 mL of LB medium supplemented with kanamycin and cultured overnight. The following day, the bacterial culture was harvested by centrifugation at 10,000 × g for 1 minute. The resulting cell pellet was resuspended in PBS and lysed via sonication (150 W, with a cycle of 1 second on and 3 seconds off, for a total of 20 minutes). The cell lysate was then transferred to a new centrifuge tube to serve as the intracellular content sample.

The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 ratio and denatured by boiling for 15 minutes. The proteins were separated on a 12% SDS-PAGE gel at 120 V. Subsequently, the proteins were transferred from the gel to a PVDF membrane under cold conditions.

The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature. To detect the His-tagged protein expressed from the pET vector, the membrane was incubated overnight at 4°C with a mouse anti-His tag primary antibody (1:1000 dilution, Cat AH367, Beyotime). The membrane was then washed three times with TBST for 10 minutes each. Following the washes, it was incubated for 1 hour at room temperature with an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat A0216, Beyotime).

After another three 10-minute washes with TBST, ECL chemiluminescent substrates A and B were mixed at a 1:1 ratio. The washed PVDF membrane was briefly placed on absorbent paper to remove excess buffer, then transferred to an imaging tray. The prepared ECL solution was applied to completely cover the membrane. After a 1-minute incubation, excess liquid was carefully wicked away, and the membrane was placed into a chemiluminescence imaging system to capture the signal according to the preset program.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img23.webp)

Figure 23: This is a flow chart of Western Blot.

## Results

Figure 24 validates the successful expression of our recombinant *TRYP* protein via Western Blot. A specific band is clearly visible at ~26 kDa in the lane corresponding to the *TRYP*-expressing strain (Lane *TRYP*). This band, which matches the predicted size of the His-tagged *TRYP*, is absent in the control lane (Lane *lacZ*). This confirms that the target protein was successfully expressed.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img24.webp)

Figure 24: SDS-PAGE analysis of recombinant *TRYP* overexpression in E. coli

## Conclusion

The Western Blot result provides unequivocal evidence for the successful expression of the recombinant *TRYP* protein(Chen et al., 2000). A distinct band appeared at the expected molecular weight of ~26 kDa exclusively in the lane corresponding to the *TRYP*-expressing strain. The absence of this band in the control lane (*lacZ*-expressing strain) confirms the high specificity of the detection. This result validates that our engineered system is capable of producing the target protein, which is the essential prerequisite for all subsequent functional assays and applications.

# The trypsin expression and refolding

## Objective

The ultimate goal of our project is to produce functional trypsin. Having successfully expressed the recombinant trypsinogen, the next critical step was to verify its catalytic activity after refolding and activation. This experiment aimed to quantify the enzymatic activity of our purified trypsin using a BAEE substrate assay and to confirm that the activity was a direct result of our engineered *TRYP* gene, by comparing it against a control strain.

## Methods

### Inclusion Body Preparation

BL21-pET-28a-*TRYP* is inoculated into 50 mL LB (100 μg/mL Kana) in 250-mL flasks and cultured at 37°C, 150 rpm.When the OD₆₀₀ of the bacteria reaches 0.6, 0.5 mM IPTG is added and the temperature is shifted to 16°C for 20-hour induction. Cells are harvested by centrifugation (10,000 × g, 5 min), resuspended in ice-cold PBS (pH 7.5), and sonicated (70W, 3-s pulse/1-s interval, 20 min; Jingxin, Shanghai). Inclusion bodies are collected by centrifugation (10,000 × g, 30 min, 4°C).

### --Inclusion Body Refolding

The pellets are washed with PBS containing 2M urea (A68476, Innochem) and incubated at 25°C, 150 rpm for 1 h. Following centrifugation (10,000 × g, 30 min, 4°C), the pellets are solubilized in denaturation buffer (20 mM Tris, 8 M urea, 20 mM DTT, pH 8.5; 10 mL/g pellet) and incubated at 25°C for 4 h with shaking. Subsequently, the supernatant is collected (12,000 × g, 20 min) and diluted 1:40 in refolding buffer (20 mM Tris, 1 mM cystine, 3 mM cysteine, 1 M urea, pH 9), after which the solution is stirred at 4°C overnight.

### --Zymogen Activation

Refolded *TRYP* is concentrated using a 10-kDa ultrafiltration tube (Millipore), resuspended in Tris buffer (40 mM Tris, 0.1 M NaCl, 10 mM CaCl₂, pH 8.0), and activated at 25°C for 1 h. Protein concentration is determined by Bradford assay (P0006, Beyotime). Samples are aliquoted and stored at -80°C.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img25.webp)

Figure 25: This is a flow chart of "Trypsin Expression and Refolding".

### Trypsin Activity Assay

The experiment is carried out in accordance with the instructions of the Trypsin (TRY) Activity Detection Kit (AKPR005M, boxbio). Trypsin can catalyze the hydrolysis of the ester bond in N-Benzoyl-L-Arginine-Ethylester (BAEE), generating N-Benzoyl-L-Arginine (BA), which has a characteristic absorption peak at 253 nm.

The operation process is as follows:

Intracellular proteins of BL21 (DE3) and BL21-*TRYP* strains are extracted and added to the reaction system (Oppenheimer et al., 1966). First, preheat a UV spectrophotometer or microplate reader for over 30 minutes, set the wavelength to 253 nm, and calibrate to zero using distilled water. Next, prewarm the detection working solution at 25°C for at least 10 minutes before testing. Then, add reagents sequentially to a 96-well UV plate or micro quartz cuvette: dispense 10 μL of crude enzyme solution into the assay group and 10 μL of distilled water into the blank group, with 200 μL of detection working solution added to both groups. For absorbance measurement: mix thoroughly, start timing immediately, and record the absorbance at 253 nm at 10 seconds (total elapsed time) as A1 (for both assay and blank groups). Incubate at 25°C for exactly 60 seconds, then measure the absorbance at 70 seconds (total elapsed time) as A2 (for both groups). Calculate ΔA for the assay group (ΔAassay = A2assay − A1assay) and the blank group (ΔAblank = A2blank − A1blank), then determine the net ΔA as ΔAassay − ΔAblank. The definition of the enzyme activity unit is: At 25 °C, per mg of protein in a 1 mL reaction system, an increase in the absorbance at 253 nm by 0.0005 per minute is defined as 1 enzyme activity unit (U/mg).

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img26.webp)

Figure 26: This is a flow chart of "Trypsin Activity Assay".

## Results

The absorbance at 253 nm of the intracellular protein of the BL21 (DE3) strain is near zero, and the corresponding enzyme activity is extremely low. the absorbance at 253 nm of the intracellular protein of the BL21-*TRYP* strain increased significantly, and the enzyme activity reached 1748 U/mg(Figure 27). Compared with the BL21 (DE3) control group, the trypsin activity of the BL21- *TRYP* strain is greatly improved, and there is an order-of-magnitude difference in absorbance and enzyme activity.

![Results Diagram](https://static.igem.wiki/teams/5986/results/img27.webp)

Figure 27 : Comparison of BAEE cleavage activity of intracellular proteins between BL21 (DE3) and *TRYP*-overexpressing strains

## Conclusion

The results provide a powerful demonstration of the functionality of our engineered trypsin. The purified protein from the BL21-*TRYP* strain exhibited high catalytic activity, reaching a specific activity of 1748 U/mg. In stark contrast, the control strain showed negligible activity, confirming that the proteolytic function is derived specifically from our expressed *TRYP* part (Rinderknecht et al., 1984).

This successful production of a highly active protease from a recombinant source validates our entire workflow, from gene design and expression to protein refolding and activation(Szilagyi et al., 2001). It establishes a solid foundation for the practical application of our enzyme in improving protein digestibility in infant formula.

# Summary

In summary, we have successfully developed and validated a robust system for producing highly active recombinant trypsin. Our key achievements are:

Successful Gene Cloning & Protein Expression: We successfully cloned the *TRYP* gene into an expression vector and confirmed its expression in *E. coli* at the protein level using Western Blot analysis. This validated our genetic design and established a reliable production strain.

Optimized Protein Processing: We established an effective protocol to overcome the challenge of inclusion body formation, which is common for heterologously expressed proteins. Our workflow for denaturation, refolding, and autocatalytic activation proved successful in converting the non-functional protein aggregates into active enzymes.

High Catalytic Activity: The final, purified trypsin demonstrated high functionality, with a specific activity of 1748 U/mg in a BAEE hydrolysis assay. This is a powerful confirmation that our entire process, from the initial DNA sequence to the final folded protein, was successful(Guy et al., 1978).

This complete, validated workflow provides a solid foundation for our project's goal: using this engineered enzyme to pre-hydrolyze casein and improve the digestibility of infant formula(Sampson et al., 1991). It also serves as a valuable case study for future iGEM teams on the production of active proteases in *E. coli*.

# System 3: *lacZ*

To address the widespread issue of lactose intolerance, this section details our successful engineering of a system for producing functional β-galactosidase (*LacZ*). Our work followed a systematic, three-step validation process: (1) Vector Construction, where we successfully cloned the codon-optimized *lacZ* gene into a pET-28a expression vector; (2) Protein Expression Verification, where we confirmed the production of the full-length, ~116 kDa *lacZ* protein in *E. coli* using Western Blot analysis; and (3) Functional Validation, where we quantified the catalytic activity of the purified enzyme *in vitro*. This comprehensive workflow demonstrates a complete "gene-to-function" pipeline for producing active lactase for potential applications in the food industry (Saqib et al., 2017).

## Construction of *lacZ* Expression Vector

## Objective

To address lactose intolerance, our goal is to produce recombinant β-galactosidase (*lacZ*). The first step is to construct the expression vector that will serve as the tool for this production. This experiment aimed to clone the codon-optimized *lacZ* gene into the pET-28a plasmid and to verify the successful construction of the resulting pET-28a-*lacZ* vector. (Bhatia et al., 2002).

## Methods

### The construction of *lacZ*-engineered bacteria

We codon optimized the open reading frame (ORF) of *lacZ* to meet RFC10 standards, synthesized it and inserted it into the pET-28a plasmid digested by NdeI and XhoI to generate the PET-28A-*lacZ* recombinant vector. The vector was transformed into E.coli BL21 (DE3) (D1009S, Beyotime, China) strain. Positive clones were screened in LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), and sequencing verification was performed (Beijing, Qingke) to obtain recombinant engineered strains. The strain was stored at -80°C with 25% (v/v) glycerol as the antifreeze. In this experiment, the culture conditions of our engineered strain were 37°C, 150 rpm, and LB liquid medium containing 100 μg/mL kana was used for inoculation and expanded culture.

![Methods Diagram](https://static.igem.wiki/teams/5986/results/img28.webp)

Figure 28: Construction of BL21-*LacZ*. (A) The plasmid map of pET28a-*LacZ*; (B) The gene circuit of BL21-*LacZ*; (C)The flow chart of Construction of BL21-*LacZ*.

## Result

As shown in Figure 29, there is a distinct band in the *lacZ* lane at approximately 3072 bp, which corresponds to the expected size of the *lacZ* gene fragment. This results demonstrate that the *lacZ* gene was successfully amplified, yielding a specific product of the expected size.

![Result Diagram](https://static.igem.wiki/teams/5986/results/img29.webp)

Figure 29: the agarose gel electrophoresis analysis of the *lacZ* gene fragment

# Conclusion

The agarose gel electrophoresis result confirms the successful construction of the pET-28a-*lacZ* expression vector. A single, clear band was observed at the expected size of 3072 bp, which perfectly matches the length of the synthesized *lacZ* gene. This result validates that the *lacZ* gene was correctly cloned into the expression plasmid, using standard molecular biology techniques (Sambrook & Russell, 2001), providing us with the essential tool needed to proceed with bacterial transformation and subsequent protein expression studies.

## Verification of Recombinant *lacZ* Protein Expression

## Objective

Having successfully constructed the pET-28a-*lacZ* vector, the next crucial step was to confirm the actual expression of the recombinant *lacZ* protein within the host cells. This experiment utilized Western Blot analysis, a highly specific immunodetection technique, to verify the presence and correct molecular weight of the His-tagged *lacZ* protein produced by our engineered *E. coli* BL21 strain.

## Methods

## Western Blot

We analyzed the *lacZ* expression by using Western blot. We inoculated engineered bacteria overexpressing *lacZ* into 50 mL LB medium thtat contain kanamycin. After overnight culture, cells were harvested by centrifugation at 10,000×g for 1 min, resuspended in PBS, and lysed by ultrasonication (150 W, 1 s on/3 s off, 20 min total). The lysate was collected as intracellular protein. Protein samples were mixed with 5× loading buffer (4:1, v/v), denatured at 100 °C for 15 min, and separated on 12% SDS-PAGE at 120 V. Proteins were transferred onto a PVDF membrane under cold conditions and blocked with 5% skim milk (TBST) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with mouse anti-His antibody (1:1000, AH367, Biyuntian), followed by HRP-conjugated goat anti-mouse IgG (1:1000, A0216, Biyuntian) for 1 h at room temperature. After each incubation, the membrane was washed three times with TBST (10 min each). Chemiluminescence was detected using ECL reagent (1:1, solutions A and B) and imaged with a chemiluminescence detection system.

![Western Blot Diagram](https://static.igem.wiki/teams/5986/results/img30.webp)

Figure 30: This is a flow chart of Western Blot.

## Result

Based on the SDS-PAGE analysis, a distinct protein band was detected in the experimental lane, migrating at the expected molecular weight (116 kDa) corresponding to β-galactosidase(Figure 31). The results verify successful expression of the *lacZ*-encoded protein, consistent with the expected molecular weight of β-galactosidase. These findings confirm that the recombinant construct enabled efficient production of the target protein.

![Result Diagram](https://static.igem.wiki/teams/5986/results/img31.webp)

Figure 31: SDS-PAGE analysis of recombinant *lacZ* overexpression in E. coli

# Conclusion

The Western Blot analysis provides unequivocal proof of successful recombinant *lacZ* expression. As shown in the results, a single, prominent band was detected at the expected molecular weight of ~116 kDa exclusively in the lane containing lysate from the *lacZ*-engineered strain (Lane 1). The absence of this band in the control lane (Lane 2, containing *TRYP* lysate) confirms the high specificity of our detection method. This result validates that our genetic construct is functional and capable of producing the full-length target protein, setting the stage for subsequent enzymatic activity assays(Zhan et al., 2014).

## Functional Assay of Purified Recombinant *lacZ*

## Objective

Having confirmed the successful expression of the *lacZ* protein, the final and most critical step was to quantify its enzymatic activity. This experiment aimed to measure the rate of lactose hydrolysis by our purified recombinant β-galactosidase *in vitro*. By monitoring the decrease in lactose concentration over an 8-hour period, we sought to provide direct, quantitative evidence of the enzyme's functionality(Zhang et al., 2020)..

## Methods

## Measurement of the lactose content *lacZ* breaks down

The purified *lacZ* enzyme with a final concentration of 0.1 U/mL was added to 1 mL of the lactose reaction system with an initial concentration of 10 mM, incubated at an appropriate temperature, and samples were taken at 0, 2, 4, 6, and 8 hours respectively. The Glucose production was determined by the Abcam Glucose Assay Kit (ab272532) to indirectly reflect lactose consumption. The specific operation is as follows: Add 5 μL of glucose standard or sample to be tested respectively to 1.5 mL centrifuge tubes, then add 500 μL of detection reagent and vortex to mix well. After tightly closing the tube cap, precisely heat in a boiling water bath for 8 minutes, and then cool in an ice water bath for 4 minutes. Take 200 μL of the reaction solution and transfer it to a 96-well plate. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 630 nm. Calculate the glucose concentration based on the standard curve and then infer the degradation amount of lactose.

![Measurement of the lactose content <em>lacZ</em> breaks down Diagram](https://static.igem.wiki/teams/5986/results/img32.webp)

Figure 32: This is a flow chart of measurement of the lactose content *lacZ* breaks down

## Result

The temporal profile of lactose concentration was monitored over an 8-hour period (Figure 33). The initial lactose concentration at 0 h was 10 mM. A gradual decline was observed with time, decreasing to 9.0 ± 0.6 mM at 2 h, followed by a more pronounced reduction to 6.5 ± 0.8 mM at 4 h. By 6 h, the lactose concentration further decreased to 4.0 ± 0.6 mM, and reached its lowest value of 2.0 ± 0.4 mM at 8 h.

![Result Diagram](https://static.igem.wiki/teams/5986/results/img33.webp)

Figure 33: Efficacy of recombinant β-galactosidase in lactose degradation.

# Conclusion

The results clearly demonstrate that our engineered and purified *lacZ* protein is highly active and functional. The enzyme effectively catalyzed the hydrolysis of lactose, reducing its concentration from 10 mM to approximately 2 mM over 8 hours, which corresponds to an 80% degradation of the initial substrate. This successful *in vitro* functional validation confirms that our recombinant *lacZ* is a viable candidate for its intended application: breaking down lactose in dairy products to address lactose intolerance(Urrutia et al., 2013).

# Summary

In summary, we have successfully constructed and validated an effective system for producing active β-galactosidase. The key milestones achieved are:

Successful Gene Cloning and Protein Expression: We first verified the correct construction of our pET-28a-*lacZ* plasmid via gel electrophoresis. Subsequently, Western Blot analysis confirmed the high-level expression of the full-length *lacZ* protein (~116 kDa) in our engineered *E. coli* strain.

Demonstrated High Catalytic Activity: The most critical finding was the functional validation of our purified enzyme. The *in vitro* assay showed that our recombinant *lacZ* is highly active, capable of degrading 80% of the lactose in the reaction system within 8 hours.

This complete and successful characterization of the existing part BBa\_I732005 not only provides new and valuable data for the iGEM community but also establishes a robust foundation for our project's application. We have produced a viable enzymatic solution with the potential to be used in creating lactose-free dairy products, directly addressing the nutritional challenges faced by individuals with lactose intolerance(Füreder et al., 2020).

# Reference

1. Bhatia, Y., Mishra, S., & Bisaria, V. S. (2002). Microbial β-Glucosidases: Cloning, Properties, and Applications. Crit. Rev. Biotechnol., 22, 375–407.
2. Buck FF, Vithayathil AJ, Bier M and Nord FF (1962). On the mechanism of enzyme action. LXXIII. Studies on trypsins from beef, sheep and pig pancreas. Archives of Biochemistry and Biophysics, 97, 417–424.
3. Chen, J.M. et al. (2000). Genes, cloned cDNAs, and proteins of human trypsinogens and pancreatitis-associated cationic trypsinogen mutations. Pancreas.
4. Chen, Y., Zhu, Y., Wang, H., Chen, R., Du, Z., & Mu, W. (2024). Developing a Metabolic Flux Optimized Escherichia coli for Efficient Production of Difucosyllactose. ACS Sustainable Chemistry & Engineering, 12(38), 14249-14257.
5. Füreder, V., Rodriguez-Colinas, B., Cervantes, F.V., Fernandez-Arrojo, L., & Plou, F.J. (2020). Selective Synthesis of Galactooligosaccharides Containing β(1→3) Linkages with β-Galactosidase from Bifidobacterium bifidum (Saphera). J. Agric. Food Chem., 68, 4930–4938.
6. Gao, F., Shi, S., Zhao, Y., Yang, D., & Liao, X. (2025). Intersecting precision fermentation for global cell-based food production innovation: Challenges and opportunities. Biotechnology Advances, 391, 108712.
7. Guy, O. et al. (1978). Two human trypsinogens. Purification, molecular properties, and N-terminal sequences. Biochemistry.
8. Jakobsson I, Lothe L, Ley D and Borschel MW (2000). Effectiveness of casein hydrolysate feedings in infants with colic. Acta Paediatrica, 89, 18–21.
9. Li, M., Zhang, T., Li, C., Jia, R., & Miao, M. (2024). Iterative multimodule reprogramming and protein engineering for efficient lactodifucotetraose bioproduction. Food Bioscience, 62, 105364.
10. Li, Y., Wang, X., Chen, K., Zhuang, Z., Tang, H., Yu, T., & Cao, W. (2025). Efficient production of 2′-fucosyllactose in Pichia pastoris through metabolic engineering and constructing an orthogonal energy supply system. Synthetic and Systems Biotechnology, 10(3), 807-815.
11. Liang, S., Quan, Q., Liu, D., Yang, S., Yan, Q., & Jiang, Z. (2025). Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in Escherichia coli. Journal of Agricultural and Food Chemistry, 73(1), 727-734.
12. Moriyama, S., Sugita, T., & Yamashita, M. (2025). Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli. Biotechnology Progress, 41(3).
13. Nyaruhucha, C.N. et al. (1997). Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. J. Biol. Chem.
14. Oppenheimer, H.L. et al. (1966). Implication of an ionizing group in the control of conformation and activity of chymotrypsin. J. Biol. Chem.
15. Rawlings, N.D. et al. (1994). Families of serine peptidases. Methods Enzymol.
16. Rinderknecht, H. et al. (1984). Mesotrypsin: a new inhibitor-resistant protease from a zymogen in human pancreatic tissue and fluid. Gastroenterology.
17. Sambrook J., Russell D. W. (2001). Molecular Cloning: A Laboratory Manual. 3rd edn. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.
18. Sampson HA, Bernhisel‐Braodbent J, Yang E and Scanlon SM (1991). Safety of casein hydrolysate formula in children with cow milk allergy. Journal of Pediatrics, 118, 520–525.
19. Saqib, S., Akram, A., Halim, S.A., & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. Biotech., 7, 79.
20. Szilagyi, L. et al. (2001). Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis. J. Biol. Chem.
21. Urrutia, P., Mateo, C., Guisan, J.M., Wilson, L., & Illanes, A. (2013). Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation. Biochem. Eng. J., 77, 41–48.
22. Yang, Y., Jing, S., Zhang, L., Shao, J., Chen, J., Xia, G., Yang, H., & Zhou, C. (2025). Structure, function, synthesis and improved strategies of fucosylated human milk oligosaccharides and their future perspectives: A review. Food Bioscience, 68, 106584.
23. Young, C. L., & Robinson, A. S. (2014). Protein Folding and Secretion: Mechanistic Insights Advancing Recombinant Protein Production in S. C. Curr. Opin. Biotechnol., 30, 168–177.
24. Zhan, Y., Xu, Z., Li, S., Liu, X., Xu, L., Feng, X., & Xu, H. (2014). Coexpression of β-d-Galactosidase and l-Arabinose Isomerase in the Production of d-Tagatose: A Functional Sweetener. J. Agric. Food Chem., 62, 2412–2417.
25. Zhang, G., Zabed, H.M., Yun, J., Yuan, J., & Qi, X. (2020). Two-stage biosynthesis of d-tagatose from milk whey powder by an engineered Escherichia coli strain expressing l-arabinose isomerase from Lactobacillus plantarum. Bioresour. Technol., 305, 123010.
26. Zhao, S., Xie, X., Yang, Y., Yu, H., Wang, J., Luo, L., & Zhou, X. (2025). Intelligent Design of Microbial Carbohydrate Factories: Integrated Chassis-Tools-Strategies Frameworks. Journal of Agricultural and Food Chemistry, 73(33), 20580-20599.

{% endblock %}