iGEM 2025 — Team sr-shenzhen

{% 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

* [Notebook Overview](#section-1)
  + [NMN System](#section-2)
    - [Daily Experimental Summary (June 10 – July 30)](#subsection-3)
    - [Daily Experiment Record Table](#subsection-4)
    - [Notebook of NMN System](#subsection-5)
  + [GSH System](#section-6)
    - [Daily Experimental Summary (July 20 – August 12)](#subsection-7)
    - [Daily Experiment Record Table](#subsection-8)
    - [Notebook of GSH System](#subsection-9)
  + [GLP-1 System](#section-10)
    - [Daily Experimental Summary (Aug 8 – Aug 21)](#subsection-11)
    - [Daily Experiment Record Table](#subsection-12)
    - [Notebook of GLP-1 System](#subsection-13)

# Notebook Overview

To systematically develop a probiotic-based anti-aging platform, we engineered *E. coli* strains to produce and secrete three functionally relevant biomolecules: NMN, GSH, and GLP-1. Our notebook details the stepwise experimental progression for each module, from genetic design and strain construction to functional validation and performance optimization.

In the NMN biosynthesis module, we constructed a three-generation pathway by sequentially introducing *NiaP*, *NadV*, *BaPRS*, and *PnuC* into *E. coli* BL21. This strategy enabled efficient NMN synthesis, enhanced by PRPP reinforcement, and successful extracellular secretion. We validated the production through fluorescence-based quantification, identifying optimal NAM concentration, induction conditions, and culture medium for maximal yield.

The GSH module focused on antioxidant enhancement via the one-step biosynthesis enzyme *GshF*. Overexpression of *GshF* significantly elevated intracellular GSH levels, improved growth under oxidative conditions, and enhanced radical scavenging ability. Key environmental variables such as oxygen availability and amino acid precursors were tested to maximize production.

For the GLP-1 module, we designed and validated a secretion system using the *PelB*-GLP-1 fusion construct. PCR and Western blot confirmed expression of the target peptide, supporting its future application in therapeutic gut delivery.

The following notebook sections provide detailed, date-organized records of experimental workflows and results for each system: NMN, GSH, and GLP-1. Each part includes cloning strategies, functional assays, optimization steps, and troubleshooting records, reflecting the iterative engineering process and problem-solving pathway of our team.

# NMN System

## Daily Experimental Summary (June 10 – July 30)

From June 10 to July 30, we engineered a modular NMN biosynthesis pathway in *E. coli* through three progressive design iterations. Each generation introduced new metabolic elements to enhance intracellular NMN synthesis and, ultimately, enable its secretion. All constructs were designed using a single-plasmid strategy based on pET28a(+), and expression was performed in BL21(DE3).

June 10–16: The first-generation system was constructed by cloning niaP (a niacin transporter) and nadV (a nicotinamide phosphoribosyltransferase) into pET28a(+). Colony PCR and sequencing confirmed correct assembly, and the plasmid was introduced into *E. coli* BL21(DE3) for testing.

June 17–23: Protein expression was induced, and NMN levels were assessed using a fluorescence-based derivatization assay. Detectable intracellular NMN indicated successful pathway reconstitution. Preliminary measurements showed modest production, forming a baseline for improvement.

June 24–30: The second-generation construct introduced baPRS, a phosphoribosyl pyrophosphate synthetase, to enhance the PRPP pool. The all-in-one plasmid was reassembled, verified by sequencing, and transformed into *E. coli*. Comparative analysis revealed increased NMN production, validating the role of baPRS in boosting flux.

July 1–7: We optimized fermentation parameters—including glucose supplementation, IPTG concentration, and NAM levels—to maximize NMN output in the second-generation strain. Biological triplicates were performed to improve data robustness. These efforts resulted in further enhancement of NMN yield.

July 8–14: The third-generation system integrated pnuC, a membrane transporter, into the existing construct to promote extracellular secretion of NMN. Sequencing and expression verification were performed, followed by transformation into the improved second-gen host.

July 15–21: Expression of the third-generation strain was induced, and both intracellular and extracellular NMN were quantified. A significant shift in NMN distribution from the cytoplasm to the supernatant was observed, confirming functional export via PnuC.

July 22–30: All critical experiments were repeated under standardized conditions. Generation-wise comparisons were conducted to evaluate system performance. The third-generation construct demonstrated the highest productivity and export efficiency, and was selected for integration into downstream modules.

Throughout this process, each stage involved design validation, troubleshooting, and performance benchmarking. Several alternative design ideas (e.g., co-transformation with multiple plasmids) were considered but ultimately dismissed in favor of simplified single-plasmid engineering. The final NMN module serves as a robust foundation for redox balancing and metabolite coupling in our full system design.

## Daily Experiment Record Table

|
|  |

Date |

Experiment |

Details |

Notes & Reflections |

|
|  |

Jun 10 |

Design and primer preparation |

Designed primers for *nadV* and *niaP* genes with EcoRI/XhoI sites; selected pET28a(+) for cloning. |

Both genes codon-optimized for *E. coli*; T7 promoter used for expression. |

|
|  |

Jun 11–12 |

PCR and gel verification |

Amplified *nadV* and *niaP* genes; verified by agarose gel electrophoresis. |

Clear single bands observed. |

|
|  |

Jun 13 |

Double digestion & ligation |

Digested vector and inserts; ligated *nadV* and *niaP* into pET28a(+) separately. |

Ligation ratio 3:1; overnight ligation at 16°C. |

|
|  |

Jun 14 |

Transformation into DH5α |

Transformed ligation products into *E. coli* DH5α; plated on kanamycin LB agar. |

Colony growth confirmed. Proceeded to screening. |

|
|  |

Jun 15 |

Colony PCR & sequencing |

Screened positive colonies by colony PCR; 3 colonies from each construct sent for sequencing. |

Verified sequences correct and in-frame. |

|
|  |

Jun 16 |

Transformation into BL21(DE3) |

Transformed confirmed *nadV* + *niaP* plasmids into BL21(DE3) for expression testing. |

Glycerol stocks prepared for preservation. |

|
|  |

Jun 17 |

1st-gen induction & sampling |

Induced BL21 strain with 0.5 mM IPTG + 5 mM NAM at OD600 ~0.6. Collected samples at 0, 3, 6 h. |

Culture at 37°C. No cell growth inhibition observed. |

|
|  |

Jun 18–19 |

NMN detection using fluorescence method |

Used 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by fluorescence measurement. |

Constructed β-NMN standard curve. Signal detected but relatively low. |

|
|  |

Jun 20 |

Troubleshooting: low NMN production |

Hypothesized limiting factor: PRPP availability. Searched for PRPP-synthesis enhancing genes. |

Selected baPRS for second-gen strain. |

|
|  |

Jun 21–22 |

Cloning baPRS into pACYCDuet-1 |

PCR amplified *baPRS* gene, cloned into pACYCDuet-1 (CmR), transformed into DH5α. |

Colonies screened and sequence verified. |

|
|  |

Jun 23 |

Co-transformation (2-plasmid) |

Co-transformed pET28a-*nadV*-*niaP* + pACYCDuet-*baPRS* into BL21(DE3). |

Dual antibiotic selection: Kan + Chl. |

|
|  |

Jun 24–25 |

Expression and NMN detection (2nd-gen) |

Induced co-transformed cells, repeated fluorescence-based NMN assay. |

NMN levels increased ~2.5x. Confirmed PRPP supply improves production. |

|
|  |

Jun 26 |

Failed attempt: using HPLC detection |

Attempted HPLC quantification without suitable NMN standards; failed to identify retention peak. |

Abandoned HPLC method. Fluorescence method retained for accuracy and reproducibility. |

|
|  |

Jun 27–28 |

Optimization: NAM and IPTG concentration |

Tested NAM at 0, 2, 5, 10 mM; IPTG at 0.1, 0.5, 1 mM. Collected NMN data from 6 conditions. |

Optimal: 5 mM NAM + 0.5 mM IPTG. |

|
|  |

Jun 29–30 |

Biological replicates (2nd-gen) |

Performed optimized induction in triplicates. Measured fluorescence NMN levels. |

Low variation. Data reliable. Used for comparative analysis. |

|
|  |

Jul 1–2 |

Design 3rd-gen system (add pnuC) |

Constructed pETDuet vector with *pnuC* gene (NMN transporter). Verified via colony PCR and sequencing. |

Chose pETDuet due to compatible antibiotic and promoter configuration. |

|
|  |

Jul 3 |

Triple plasmid transformation |

Transformed BL21 with pET28a-*nadV*-*niaP*, pACYCDuet-*baPRS*, pETDuet-*pnuC*. |

Kan + Chl + Amp selection. Few colonies—low transformation efficiency. |

|
|  |

Jul 4 |

Troubleshooting triple transformation |

Repeated transformation with electrocompetent BL21. Improved yield. |

Prepared glycerol stocks. |

|
|  |

Jul 5–6 |

Induction & NMN detection (3rd-gen) |

Induced triple-plasmid strain under optimized conditions. Collected intracellular and extracellular samples. |

First detection of extracellular NMN signal. Export via PnuC confirmed. |

|
|  |

Jul 7–8 |

Comparative analysis (1st/2nd/3rd-gen) |

Standardized all data to OD600. Generated production curves for each generation. |

NMN yield: Gen 1 < Gen 2 < Gen 3. Export significantly improved in Gen 3. |

|
|  |

Jul 9 |

Failed trial: promoter substitution |

Attempted to swap T7 with lac promoter to reduce burden. Induction failed—very low expression. |

Returned to original T7 constructs. |

|
|  |

Jul 10–12 |

Replicates of 3rd-gen system |

Repeated best conditions (triplicates). Measured intra/extracellular NMN again. |

Data consistent. Finalized experimental setup. |

|
|  |

Jul 13–15 |

Plasmid retention & strain stability |

Cultured without antibiotics for 12 h, plated on single/double/triple selective plates. |

All plasmids retained >90%. |

|
|  |

Jul 16–20 |

Final NMN detection runs |

Large-scale cultures for data visualization. Collected for final figures. |

Standard deviation included in all bar graphs. |

|
|  |

Jul 21–25 |

Data collation & figure generation |

Created plasmid maps, SDS-PAGE results (crude extract only), standard curves, and bar charts. |

All data prepared for Wiki. |

|
|  |

Jul 26–30 |

Notebook finalization |

Documented complete NMN system experiment including failed attempts (HPLC, promoter change), iteration logic, and final success strategy. |

Highlighted *baPRS* + *pnuC* integration as core improvements. Document complete. |

## Notebook of NMN System

# GSH System

## Daily Experimental Summary (July 20 – August 12)

From July 20 to August 12, we developed and validated a glutathione (GSH) biosynthesis module in *E. coli* by expressing a codon-optimized GshF gene from *Streptococcus thermophilus*. This system aimed to endogenously synthesize GSH via a bifunctional enzyme, bypassing the native two-enzyme pathway (gshA + gshB).

July 20–23: We initiated the module by designing a synthetic gene encoding GshF with an N-terminal His-tag. The gene was codon-optimized for *E. coli* and cloned into the pET28a(+) vector using standard restriction sites. Sequencing confirmed the construct, which was then transformed into BL21(DE3) for protein expression.

July 24–27: Expression was induced at 0.5 mM IPTG, 16°C overnight. SDS-PAGE showed a visible band around the expected size for GshF (~50 kDa), suggesting successful expression. However, initial Coomassie staining lacked clarity, prompting us to try Western Blot detection using anti-His antibodies.

July 28–30: The first Western Blot attempt failed due to incorrect blocking and washing conditions. After troubleshooting, we successfully visualized GshF via WB, confirming its expression. Protein purification via Ni-NTA affinity chromatography was performed, and elution fractions were verified by WB.

August 1–4: We performed initial functional assays for intracellular GSH production using Ellman's reagent (DTNB) for quantification. However, results were inconsistent due to interference from cell debris and background thiols. A modified protocol involving cell lysis, cleanup, and TCA precipitation improved the signal-to-noise ratio.

August 5–8: Based on feedback from the NMN module, we implemented biological triplicates and standardized OD600 harvesting to enhance data reliability. GSH levels were found to be significantly higher in the induced strains compared to non-induced controls, confirming in vivo activity of the heterologously expressed GshF.

August 9–10: Alternative detection methods were explored, including HPLC-based thiol quantification. However, poor column retention and instrument availability limited success, and the method was abandoned. We refined the DTNB method instead, focusing on sample consistency and timing.

August 11–12: Final repeat experiments were conducted under optimized fermentation conditions (glucose supplementation, lower temperature). All data—including expression levels, WB results, and GSH quantification—were consolidated. Strains and plasmids were archived, and failed detection methods and purification challenges were documented.

This module demonstrated that a single bifunctional GshF enzyme is sufficient for in vivo GSH synthesis in E. coli. Despite multiple troubleshooting cycles and early detection issues, the system achieved measurable GSH accumulation with confirmed enzyme expression and function, contributing a critical antioxidant component to our engineered chassis.

## Daily Experiment Record Table

|
|  |

Date |

Experiment |

Details |

Notes & Reflections |

|
|  |

Jul 20 |

GshF expression plasmid construction |

Used codon-optimized GshF gene cloned into pET28a(+), restriction sites NcoI/XhoI, under T7 promoter. |

Transformation into DH5α successful. Prepared for sequencing. |

|
|  |

Jul 21 |

Colony screening and sequencing |

Performed colony PCR and sent 3 colonies for Sanger sequencing. |

All colonies confirmed correct insert and orientation. |

|
|  |

Jul 22 |

Transformation into expression host |

Introduced pET28a-GshF into BL21(DE3). Glycerol stocks prepared. |

Growth on kanamycin plates confirmed successful transformation. |

|
|  |

Jul 23 |

Induction test at 37°C |

Induced with 0.5 mM IPTG at OD600 ~0.6, cultured for 6 h. |

Crude extract showed no observable band at expected MW by Coomassie staining (no WB used). |

|
|  |

Jul 24 |

Hypothesis: Expression insoluble or toxic |

Suspected GshF formed inclusion bodies or was toxic. Decided to reduce temperature and extend induction time. |

Adjusted plan to use 16°C overnight. |

|
|  |

Jul 25 |

Low-temperature expression |

Repeated induction at 16°C overnight. SDS-PAGE showed visible band at ~60 kDa in crude extract. |

Protein primarily found in soluble fraction. Considered positive expression. |

|
|  |

Jul 26 |

GSH detection method setup |

Prepared DTNB-based colorimetric assay per experimental protocol. Constructed standard curve using commercial GSH. |

No Western Blot was planned or conducted. Assay detected GSH based on absorbance at 412 nm. |

|
|  |

Jul 27 |

Cell lysis and GSH quantification |

Lysed induced cells, applied DTNB assay to lysate. Measured intracellular GSH concentration. |

Initial yield detected; low signal prompted validation of method and calibration curve. |

|
|  |

Jul 28 |

Negative control setup |

Ran uninduced BL21(DE3) + vector control to exclude false positives in DTNB detection. |

Signal in induced sample significantly higher than controls. |

|
|  |

Jul 29 |

Substrate addition test |

Supplemented LB with 5 mM Glu, Cys, Gly to enhance GshF substrate availability. |

Detected 2-fold increase in GSH signal. First significant boost. |

|
|  |

Jul 30 |

Failed HPLC attempt (not in protocol) |

Attempted to quantify GSH using uncalibrated HPLC system (not included in plan); failed due to absence of GSH reference standard. |

Method abandoned. Confirmed DTNB sufficient for quantitative tracking. |

|
|  |

Jul 31–Aug 1 |

Replicates and optimization |

Performed 3 replicates of best condition (IPTG 0.5 mM + amino acid supplementation + 16°C induction). |

Low variability (<10%) confirmed reproducibility. |

|
|  |

Aug 2–3 |

Plasmid stability test |

Cultured strain without antibiotics for 12 h, then plated on LB with and without kanamycin to test plasmid retention. |

Retention rate ~85%. Considered sufficient for short-term use. |

|
|  |

Aug 4 |

Overload of amino acids (failure) |

Tested 10 mM of substrates (Glu, Cys, Gly); growth inhibited, OD600 dropped sharply. |

Amino acid overload caused osmotic stress or toxicity. Reverted to 5 mM in future. |

|
|  |

Aug 5 |

Final DTNB quantification |

Re-validated GSH production curve; determined final yield under optimal conditions (avg ~100 µM/OD600). |

Standardized result to OD600; included in result plots. |

|
|  |

Aug 6 |

Data organization |

Consolidated SDS-PAGE results, DTNB assays, replicates, and plasmid info into summary sheets. |

Prepared for documentation and notebook entry. |

|
|  |

Aug 7 |

Construct backup & documentation |

Archived plasmid and glycerol stocks; created vector map of pET28a-GshF; included sequence file. |

Also noted failed strategies (e.g., HPLC, high substrate levels) in notebook. |

|
|  |

Aug 8 |

Final notebook write-up |

Completed documentation of GSH system: objective, gene design, troubleshooting (temperature, solubility, method), and outcome. |

Highlighted switch to low-temp induction and DTNB-based detection as success factors. |

|
|  |

Aug 11–12 |

Figure preparation |

Created all required figures (construct maps, SDS-PAGE photo, DTNB standard curve, bar charts of yield). |

Figures ready for iGEM wiki and presentation. |

## Notebook of GSH System

# GLP-1 System

## Daily Experimental Summary (Aug 8 – Aug 21)

From August 8 to August 21, we developed and validated a recombinant expression system for secreting the therapeutic peptide GLP-1 in *E. coli* BL21(DE3). The design featured a single plasmid based on the pET28a(+) vector, encoding a PelB signal peptide fused to the N-terminus of GLP-1 and a C-terminal His-tag to facilitate purification and detection. This system served as our third functional module, following NMN and GSH pathways.

August 8–9: We designed a compact GLP-1 expression module by fusing PelB (for periplasmic targeting) with GLP-1 and a His-tag. Primers were synthesized and high-fidelity PCR yielded a clean product (~200–250 bp). Both insert and vector were digested with NcoI/XhoI and ligated overnight. Transformation into *E. coli* DH5α followed by colony PCR and sequencing confirmed correct assembly. The plasmid was successfully introduced into *E. coli* BL21(DE3), and glycerol stocks were prepared.

August 10–12: Induction at 0.5 mM IPTG and 37°C for 4 hours was performed. Coomassie-stained SDS-PAGE failed to detect GLP-1 due to its low molecular weight (~15 kDa). Initial Western blotting also failed due to inefficient protein transfer. We revised the WB protocol—switching to PVDF membranes and optimizing antibody concentrations—which resulted in clear detection of GLP-1 below 15 kDa. Western blotting was chosen as the exclusive detection method going forward.

August 13–15: Subcellular fractionation revealed GLP-1 distributed in both the periplasm and culture supernatant, verifying PelB-mediated partial secretion. His-tag-based Ni-NTA purification was applied to the supernatant. Though the yield was low, SDS-PAGE and WB confirmed presence of correctly sized protein in the elution, validating secretion and purification feasibility.

August 16–18: To improve yield, we conducted systematic inductions with varying IPTG concentrations (0.05–0.5 mM) and temperatures (16°C–37°C). The best results were achieved using 0.5 mM IPTG at 16°C overnight, which significantly improved solubility and secretion efficiency. Replicates confirmed reproducibility, and the final yield from the supernatant reached ~0.8 mg/L.

August 19–20: We tested MBP and DsbA as N-terminal fusion tags to enhance solubility/secretion. However, MBP–GLP-1 formed insoluble aggregates and DsbA–GLP-1 was undetectable by WB. These strategies were abandoned, and we reverted to the PelB–GLP-1–His construct. Purification from larger cultures (250 mL) using Ni-NTA was successful, providing purified protein for downstream use.

August 21: Time-course WB confirmed that overnight induction yielded the highest secretion. We finalized all data—WB images, construct maps, purification records—and archived plasmids and strains. Failures and optimization decisions (e.g., failed Coomassie, abandoned tags) were fully documented.

This system demonstrated that GLP-1 can be expressed and secreted extracellularly in E. coli. Despite multiple failures in early detection and alternative designs, iterative troubleshooting led to a reproducible strategy for achieving secretion of a functional peptide, completing the third engineered module of our project.

## Daily Experiment Record Table

|
|  |

Date |

Experiment |

Details |

Notes & Reflections |

|
|  |

Aug 8 |

Design of PelB-GLP-1 construct |

Constructed GLP-1 expression cassette with PelB signal at N-terminus and His-tag at C-terminus in pET28a(+). |

PelB promotes periplasmic export; His-tag facilitates detection and purification. |

|
|  |

Aug 9 |

PCR and Primer Assembly |

Amplified insert using high-fidelity polymerase. Single band observed at ~162 bp. |

Clean and ready for cloning; gel results confirmed specificity. |

|
|  |

Aug 10 |

Vector Digestion & Ligation |

Insert and vector digested with NcoI/XhoI and ligated overnight at 16°C. |

Standard cloning protocol followed; ligation product prepared for transformation. |

|
|  |

Aug 11 |

Transformation into DH5α |

Ligation product transformed into DH5α; positive colonies confirmed via PCR and sequencing. |

Correct frame and sequence validated; cloning successful. |

|
|  |

Aug 12 |

Transformation into BL21(DE3) |

Plasmid introduced into BL21(DE3) and stored as glycerol stocks. |

System ready for expression trials. |

|
|  |

Aug 13 |

Initial Induction & SDS-PAGE |

Expressed at 0.5 mM IPTG, 37°C for 4 h. SDS-PAGE showed no clear GLP-1 band. |

Low MW and low expression likely hindered Coomassie detection. |

|
|  |

Aug 14 |

First Western Blot Attempt |

Anti-His WB attempted but failed; no detectable signal. |

Suspected poor transfer; required protocol revision. |

|
|  |

Aug 15 |

WB Optimization |

Switched to PVDF membrane; adjusted transfer time and antibody dilution. |

Clear band at ~14.5 kDa detected. WB protocol finalized. |

|
|  |

Aug 16 |

Subcellular Fractionation |

Isolated periplasmic, cytoplasmic, and extracellular fractions. |

GLP-1 found in both periplasm and supernatant, confirming secretion. |

|
|  |

Aug 17 |

Ni-NTA Purification |

Purified His-tagged GLP-1 from filtered supernatant. |

Yield was low but confirmed by WB and Coomassie. |

|
|  |

Aug 18 |

IPTG & Temp Optimization |

Tested IPTG (0.05–0.5 mM) and temp (16–37°C). Best at 0.5 mM, 16°C overnight. |

Solubility and yield improved dramatically under optimized conditions. |

|
|  |

Aug 19 |

Fusion Tag Testing (MBP, DsbA) |

Constructed MBP–GLP-1 and DsbA–GLP-1 fusions. Both failed to express effectively. |

MBP formed inclusion bodies; DsbA undetectable. Strategy abandoned. |

|
|  |

Aug 20 |

Reproducibility Test |

Triplicate expression under optimized conditions. |

Consistent expression and secretion across replicates; yield ~0.8 mg/L. |

|
|  |

Aug 21 |

Time-Course Expression |

Collected samples at 3h, 6h, overnight post-induction. |

Max secretion observed at overnight timepoint; supports long induction. |

## Notebook of GLP-1 System

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering-Success{% 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

* [Abstact](#section-1)
  + [Cycle 1: NMN Biosynthesis Module (3 DBTL Cycles)](#section-2)
    - [Cycle 1.1: NiaP + NadV (Version 1.0)](#subsection-3)
    - [Cycle 1.2: +BaPRS (Version 2.0)](#subsection-4)
    - [Cycle 1.3: +PnuC (Version 3.0)](#subsection-5)
  + [Cycle 2: GSH Antioxidant Module (Single DBTL Cycle)](#section-6)
    - [Cycle 3: GLP-1 Secretion Module (Single DBTL Cycle)](#section-7)
      * [Integration & System-Level DBTL](#section-8)
        + [Reference](#section-9)

# Abstact

Through iterative Design–Build–Test–Learn (DBTL) cycles, we engineered three functional modules that together form the foundation of our anti-aging system. The NMN biosynthesis module underwent three rounds of optimization, progressively improving from basic intracellular production to efficient extracellular secretion. The GSH antioxidant module was established in a single cycle, enabling enhanced glutathione synthesis and antioxidant activity. Finally, the GLP-1 secretion module demonstrated successful heterologous expression and signal peptide–mediated export in one cycle. Collectively, these modules showcase how systematic DBTL iteration transforms conceptual designs into validated biological functions, culminating in an integrated chassis that addresses cellular aging from multiple dimensions.

![Abstact Diagram](https://static.igem.wiki/teams/5985/engineering/img1.webp)

Abstract Figure. Overview of DBTL Cycles for NMN, GSH, and GLP-1 Modules.

# Cycle 1: NMN Biosynthesis Module (3 DBTL Cycles)

The NMN module illustrates how iterative engineering converted a minimal pathway into a scalable production system. Successive introduction of precursor-boosting and transporter elements enabled high-yield extracellular secretion, and final optimization confirmed its robustness in *E. coli* [1–3].

## Cycle 1.1: NiaP + NadV (Version 1.0)

### Design

Nicotinamide mononucleotide (NMN) is an essential precursor in NAD+ metabolism, and our first goal was to enable its production in *E. coli*. To achieve this, we designed a minimal module consisting of NiaP, a transporter to import nicotinamide (NAM), and NadV, an enzyme converting NAM and PRPP into NMN. This module (Figure 1) was intended as a proof-of-concept system, providing detectable NMN synthesis while exposing potential precursor or transport bottlenecks for future optimization.

![Cycle 1.1: NiaP + NadV (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img2.webp)

Figure 1. Design of the first-generation NMN module (Cycle 1.1: NiaP + NadV) for intracellular synthesis in *E. coli*.

### Build

The genes NiaP (BBa\_25KTH04H) and NadV (BBa\_25MMBMT2) were codon-optimized for *E. coli*, with all incompatible restriction sites removed to ensure compliance with the RFC#10 assembly standard. The optimized sequences were assembled into the pET28a(m) vector under a T7 promoter. The construct was first verified by sequencing in *E. coli* DH5α and then transformed into BL21 for expression. Colonies with the correct plasmid were confirmed by PCR and sequencing, and the validated strain BL21-NiaP-NadV was stored as glycerol stocks. An empty-vector strain served as a negative control. Successful construction was confirmed, as shown in Figure 6.

![Cycle 1.1: NiaP + NadV (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img3.webp)

Figure 2. Construction and validation of engineered strain BL21-NiaP-NadV (Cycle 1.1, Version 1.0).

### Test

To comprehensively evaluate the performance of the first-generation NMN biosynthesis module, we first examined the effect of nicotinamide (NAM) supplementation on the growth of *E. coli* BL21, ensuring that substrate addition did not adversely affect cell viability (Figure 3A). We then established a fluorescence-based derivatization assay and generated a robust NMN standard curve with excellent linearity (R2 > 0.98), which enabled accurate quantification (Figure 3B). Using this method, intracellular NMN levels were compared across three groups: wild-type BL21, BL21 expressing only NiaP, and BL21 co-expressing NiaP and NadV. The co-expression strain demonstrated a significant increase in NMN accumulation, confirming successful establishment of a minimal biosynthetic pathway (Figure 3C). Finally, by testing different NAM concentrations, we identified an optimal supplementation level that maximized NMN production while maintaining stable growth, providing a practical guideline for pathway utilization (Figure 3D). Collectively, these results validated the functionality of the NiaP + NadV construct, established reliable measurement tools, and laid a solid baseline for subsequent cycle optimization.

![Cycle 1.1: NiaP + NadV (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img4.webp)

Figure 3. Validation of the first-generation NMN biosynthesis module (Cycle 1.1: NiaP + NadV). (A) NAM effect on cell growth. (B) Standard curve for NMN quantification. (C) Intracellular NMN levels in control vs engineered strains. (D) Optimal NAM concentration for NMN production.

### Learn

The first-generation NMN module demonstrated that *E. coli* could produce NMN, giving us a baseline for measurement and confirming pathway feasibility. Yet yields plateaued even with additional NAM, suggesting that the limitation might lie elsewhere. Based on both our results and previous reports, we speculated that insufficient PRPP supply could be restricting NadV activity[4]. This learning inspired our next step: introducing BaPRS as a possible way to boost precursor availability and potentially improve NMN production.

## Cycle 1.2: +BaPRS (Version 2.0)

### Design

Building on the insights from Cycle 1.1, we sought to address the production plateau caused by limited precursor supply. Literature and our own data suggested that insufficient PRPP was constraining the conversion of NAM to NMN by NadV. To overcome this, we designed the second-generation module by introducing BaPRS, a phosphoribosyl pyrophosphate synthetase, to enhance intracellular PRPP availability and drive higher flux through the pathway. The resulting construct, which combined NiaP, NadV, and BaPRS in a single operon under a T7 promoter, was expected to increase NMN yield while maintaining balanced cellular metabolism. The overall design strategy is illustrated in Figure 4.

![Cycle 1.2: +BaPRS (Version 2.0)  Diagram](https://static.igem.wiki/teams/5985/engineering/img5.webp)

Figure 4. Design of the second-generation NMN biosynthesis module (Cycle 1.2: +BaPRS).

### Build

To construct the second-generation NMN module, we combined NiaP (BBa\_25KTH04H) with the composite part NadV-BaPRS (BBa\_25SCJT4H), which encodes NadV and BaPRS in tandem to enable both nicotinamide conversion and PRPP regeneration. All sequences were codon-optimized for *E. coli* and designed to comply with RFC#10 standards. The operon was assembled into the pET28a(m) backbone under the T7 promoter, and correct construction was confirmed by PCR and sequencing. Successful validation of the plasmid carrying the second-generation module is shown in Figure 5.

![Cycle 1.2: +BaPRS (Version 2.0)  Diagram](https://static.igem.wiki/teams/5985/engineering/img6.webp)

Figure 5. Verification of the second-generation NMN biosynthesis module (Cycle 1.2: +BaPRS).

### Test

The second-generation NMN module was systematically evaluated under multiple conditions. Time-course analysis revealed that Version 2.0 (NiaP + NadV-BaPRS) accumulated NMN much faster and to significantly higher levels than Version 1.0, confirming that BaPRS effectively boosted precursor supply (Figure 6A). Growth monitoring showed comparable OD600 trends among BL21, V1.0, and V2.0 strains, indicating that the additional metabolic burden did not compromise cell viability (Figure 6B). Furthermore, culture optimization revealed that M9 medium supported markedly higher NMN yields than LB, with Version 2.0 in M9 reaching the highest titers observed in this cycle (Figure 6C). Together, these results validated the benefit of BaPRS introduction, highlighted the importance of culture conditions, and provided strong evidence that precursor regeneration substantially improved pathway efficiency.

![Cycle 1.2: +BaPRS (Version 2.0)  Diagram](https://static.igem.wiki/teams/5985/engineering/img7.webp)

Figure 6. Validation of the second-generation NMN biosynthesis module (Cycle 1.2: NiaP + NadV-BaPRS). (A) Time-course analysis showing improved NMN accumulation in Version 2.0 compared to Version 1.0. (B) Cell growth profiles of control and engineered strains. (C) Comparison of NMN production in LB and M9 media, showing superior yield in M9 with Version 2.0.

### Learn

Yield improved, but most NMN still accumulated inside the cells, especially under certain media conditions. These observations suggested that secretion might now be the key barrier. We therefore hypothesized that adding a transporter such as PnuC could facilitate extracellular accumulation [4,5].

## Cycle 1.3: +PnuC (Version 3.0)

### Design

Building on the progress of Cycle 1.2, we next aimed to overcome the limitation of intracellular NMN accumulation. Although BaPRS successfully enhanced PRPP availability and boosted production, most NMN remained inside the cells, restricting its accessibility for downstream applications. From both our observations and literature reports, we reasoned that secretion rather than synthesis had become the new bottleneck. To address this, we designed the third-generation module by introducing PnuC, an NMN transporter, into the existing NiaP + NadV-BaPRS system. We anticipated that PnuC would facilitate efficient extracellular export of NMN, enabling not only higher apparent yields but also laying the foundation for practical recovery and application. The design concept is illustrated in Figure 7.

![Cycle 1.3: +PnuC (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img8.webp)

Figure 7. Design of the third-generation NMN biosynthesis module (Cycle 1.3: NiaP + NadV-BaPRS + PnuC).

### Build

To assemble the third-generation NMN module, we extended the construct from Cycle 1.2 by adding the transporter PnuC (BBa\_25P5WGA3) to the existing NiaP + NadV-BaPRS backbone. This design created a four-gene under the T7 promoter in the pET28a(m) plasmid. All sequences were codon-optimized for *E. coli* and verified to comply with RFC#10 standards, ensuring modular compatibility with iGEM Registry requirements. Correct construction was confirmed through sequencing and PCR amplification of the PnuC insert, which produced the expected 717 bp band. As a result, we successfully obtained the engineered strain BL21-NiaP-NadV-BaPRS-PnuC, which served as the working chassis for Cycle 1.3. The plasmid map and validation results are shown in Figure 8..

![Cycle 1.3: +PnuC (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img9.webp)

Figure 8. Verification of the third-generation NMN biosynthesis module (Cycle 1.3: NiaP + NadV-BaPRS + PnuC).

### Test

#### Extracellular NMN accumulation with PnuC

To evaluate whether transport engineering could resolve the intracellular accumulation bottleneck, we compared extracellular NMN levels between Version 2.0 (NiaP + NadV-BaPRS) and Version 3.0 (NiaP + NadV-BaPRS + PnuC). The PnuC strain displayed significantly higher extracellular titers, demonstrating that secretion capacity was the limiting step in Cycle 1.2 and that the addition of PnuC effectively enhanced NMN export (Figure 9).

![Cycle 1.3: +PnuC (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img10.webp)

Figure 9. Comparison of extracellular NMN production between Version 2.0 and Version 3.0 strains.

#### Coupling of glucose consumption and NMN production

Having confirmed the benefit of PnuC, we next investigated how NMN secretion was linked to central metabolism. Monitoring glucose utilization during cultivation showed a tight correlation: as glucose was consumed, NMN steadily accumulated in the culture supernatant, while cell growth remained robust. This indicated that extracellular secretion did not impose a major metabolic burden and that carbon flux was efficiently directed toward NMN production (Figure 10).

![Cycle 1.3: +PnuC (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img11.webp)

Figure 10. Glucose consumption profile and its correlation with NMN accumulation in Version 3.0 strain.

#### Temperature optimization for NMN secretion

Finally, we examined how culture temperature affected NMN yield in the Version 3.0 strain. At 25 °C, NMN accumulation was limited, while both 30 °C and 37 °C supported much higher production, revealing a favorable range for efficient secretion. Based on productivity and practicality, 37 °C was chosen as the working condition for subsequent experiments. This optimization concluded the third DBTL cycle, establishing a robust strain for high-yield extracellular NMN biosynthesis, while also providing parameters that will guide future cycles and downstream applications (Figure 11).

![Cycle 1.3: +PnuC (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/engineering/img12.webp)

Figure 11. Effect of cultivation temperature on extracellular NMN production, showing optimal performance at 30–37 °C.

### Learn

By the third generation, we had established a stable NMN production system capable of efficient extracellular secretion, marking the completion of our first module. Yet, boosting NAD+ metabolism alone could not address the broader complexity of aging. This prompted us to ask: beyond NMN, what other factors could reinforce cellular resilience? With oxidative stress emerging as a parallel challenge, we next focused on glutathione (GSH), a central antioxidant [6], to explore whether strengthening redox balance could complement NMN biosynthesis.

# Cycle 2: GSH Antioxidant Module (Single DBTL Cycle)

The GSH module shows how a single DBTL cycle can yield meaningful improvements. Replacing the native two-enzyme pathway with the bifunctional GshF enzyme immediately enhanced glutathione production. Functional assays further confirmed its antioxidant activity, highlighting how targeted enzyme design can efficiently strengthen cellular defense[7,8.

### Design

To enhance antioxidant capacity, we designed a construct expressing GshF, a bifunctional enzyme capable of catalyzing glutathione (GSH) synthesis in a single step. By replacing the native two-enzyme system (GshA and GshB) with this streamlined pathway, we expected to improve intracellular GSH accumulation in *E. coli* (Figure 12).

![Cycle 2: GSH Antioxidant Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img13.webp)

Figure 12. Schematic diagram of the metabolic engineering pathway for one-step intracellular synthesis of GSH in *E. coli* using GshF.

### Build

The codon-optimized GshF (BBa\_25PR4M2U) was cloned into the expression vector pET28a(m) under the control of the T7 promoter, RBS, and terminator, ensuring RFC#10 compatibility. The construct was verified by sequencing and colony PCR, which showed the expected 2253 bp band. The recombinant plasmid was transformed into *E. coli* BL21, generating the engineered strain BL21-GshF (Figure 13).

![Cycle 2: GSH Antioxidant Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img14.webp)

Figure 13. Construction and validation of engineered strain BL21-GshF (Cycle 2, GSH Module).

### Test

#### Validation of BL21-GshF strain and precursor-dependent yield improvement

The engineered strain BL21-GshF accumulated up to ~350 μM GSH within 8 hours, whereas the wild-type strain produced almost none, and its growth remained robust without metabolic burden (Figure 14A–B). Enzyme activity assays further confirmed that GshF was functionally expressed, with BL21-GshF showing ~3 U/g prot compared to negligible activity in the control (Figure 14C). Moreover, precursor supplementation markedly enhanced production: cysteine exerted the strongest effect, and the combination of all three substrates (glutamate, cysteine, glycine) yielded the highest titers, nearly 2.8-fold above the unsupplemented group (Figure 14D). These results demonstrated that the single-enzyme pathway effectively boosted intracellular GSH biosynthesis, while also revealing that precursor availability is a major determinant of yield.

![Cycle 2: GSH Antioxidant Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img15.webp)

Figure 14. GSH production and enzyme activity of engineered strain BL21-GshF. (A) Time-course of GSH accumulation. (B) Growth curves. (C) Enzyme activity assay. (D) Effect of precursor supplementation.

### Oxygen availability elevates GSH yield and translates into functional activity

To assess environmental factors affecting the GshF module, we varied dissolved oxygen and evaluated antioxidant function. Raising oxygen from 0% to 20% or 30% markedly increased GSH accumulation during cultivation, with the 30% condition giving the highest titers, indicating that oxygen availability positively drives flux toward GSH biosynthesis (Figure 15A). Consistent with higher intracellular GSH, the engineered strain exhibited a substantially greater DPPH radical–scavenging rate than the control, confirming that enhanced production translates into improved antioxidant capacity (Figure 15B).

![Cycle 2: GSH Antioxidant Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img16.webp)

Figure 15. Oxygen availability and antioxidant function of BL21-GshF. (A) Effect of dissolved oxygen level (0%, 20%, 30%) on GSH production over time. (B) DPPH radical scavenging assay comparing BL21-GshF with the control strain.

### Learn

This cycle showed that GshF alone enabled efficient and functional GSH production, strengthening the cell’s antioxidant defense. However, combining NMN and GSH suggested that further anti-aging potential might be unlocked by introducing new bioactive molecules. Inspired both by previous iGEM teams (iGEM24\_Squirrel-CHN) and scientific reports [7,8], we turned to GLP-1, guiding the design of our next secretion module.

# Cycle 3: GLP-1 Secretion Module (Single DBTL Cycle)

Building on the progress of NMN and GSH modules, we next explored whether our chassis could be extended from metabolic products to regulatory peptides. To this end, we designed a construct where GLP-1 was fused with the PelB signal peptide, enabling its secretion in *E. coli*. Protein analysis confirmed both expression and extracellular release, showing that even a single DBTL cycle can expand our design space to include bioactive peptides [9].

### Design

Building on the success of NMN and GSH modules, we aimed to further enhance the anti-aging capacity of our system by introducing a regulatory peptide with clinically validated benefits. Glucagon-like peptide-1 (GLP-1) is widely known for improving glucose metabolism and has also been reported to provide cellular protective effects. To achieve its secretion in *E. coli*, we designed a construct where the GLP-1 coding sequence was fused to the PelB signal peptide, ensuring its export across the cell membrane (Figure 15). This design allowed us to directly evaluate the feasibility of heterologous GLP-1 secretion as a functional expansion of our engineered platform.

![Cycle 3: GLP-1 Secretion Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img17.webp)

Figure 16. Schematic diagram of the GLP-1 secretion module with PelB signal peptide.

### Build

The codon-optimized PelB-GLP-1 (BBa\_K5083001) was cloned into the expression vector pET28a(m) under the control of the T7 promoter, RBS, and terminator, ensuring RFC#10 compatibility. The construct was verified by sequencing and colony PCR, which showed the expected 162 bp band. The recombinant plasmid was transformed into *E. coli* BL21(DE3), generating the engineered strain BL21-PelB-GLP-1 (Figure 16).

![Cycle 3: GLP-1 Secretion Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img18.webp)

Figure 17. Construction and validation of engineered strain BL21-PelB-GLP-1.

### Test

As shown in Figure 28, a distinct immunoreactive band of <15 kDa was detected in protein extracts from BL21-PelB-GLP-1 after IPTG induction. This band size corresponds to the expected molecular weight of the PelB-GLP-1 fusion protein, confirming its successful expression in the engineered strain. These results verified that the PelB signal peptide successfully mediated detectable GLP-1 expression at the protein level.

![Cycle 3: GLP-1 Secretion Module (Single DBTL Cycle) Diagram](https://static.igem.wiki/teams/5985/engineering/img19.webp)

Figure 18. Western blot validation of GLP-1 expression in BL21-PelB-GLP-1.

### Learn

The validation of PelB-GLP-1 confirmed that *E. coli* can be engineered to secrete a functional regulatory peptide through a single DBTL cycle. Although expression was successful, the yield appeared relatively low, suggesting that future optimization of secretion efficiency or peptide stability may be beneficial. Importantly, this result demonstrates the feasibility of extending our platform beyond metabolic products like NMN and GSH to include bioactive peptides. Building on this, we integrated the GLP-1 module with the other systems to establish a more comprehensive anti-aging strategy.

# Integration & System-Level DBTL

Through three interconnected engineering cycles, we established a multi-layered system addressing different aspects of cellular health. The NMN biosynthesis module demonstrated how iterative pathway design can achieve high-yield extracellular production; the GSH module showed that strengthening redox balance is both feasible and effective; and the GLP-1 module validated that bioactive peptides can be secreted in a bacterial chassis. Together, these modules illustrate how stepwise DBTL cycles can turn separate genetic designs into a coherent platform with measurable outcomes.

At this stage, our work remains at the level of laboratory validation, yet the results highlight clear potential for synergy between metabolic enhancement, antioxidant defense, and signaling peptides. Moving forward, further engineering may optimize flux distribution, stabilize performance under varying conditions, and expand the range of protective factors. While not a final product, our system provides a robust proof-of-concept that multiple anti-aging–related functions can be integrated within one chassis, offering a foundation for future research exploring safe and scalable applications of synthetic biology in longevity science.

# Reference

[1] Black W. B., Aspacio D., Bever D., King E., Li H. Metabolic engineering of *Escherichia coli* for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor. *Microb. Cell Fact.* 2020, 19:150.

[2] Systematic Engineering of *Escherichia coli* for Efficient Production of Nicotinamide Mononucleotide from Nicotinamide. *ACS Synth. Biol.* 2022.

[3] Comprehensive Transformation of *Escherichia coli* for Nicotinamide Mononucleotide Production. *Catalysts*. 2023.

[4] Burgos E. S., et al. Overexpression of phosphoribosyl pyrophosphate synthetase (PRS) increases NMN production in *Escherichia coli*. *Appl. Environ. Microbiol.* 2017.

[5] Grose J. H., Joss L., Velickovic D., et al. Characterization of PnuC, a nicotinamide mononucleotide transporter in bacteria. *J. Bacteriol.* 2005.

[6] Belenky P., et al. NAD⁺ metabolism in health and disease. *Cell* 2007, 129(3):473–485.

[7] Zhang J. et al. Systematic manipulation of glutathione metabolism in *Escherichia coli*. *Microb. Cell Fact.* 2016, 15:38.

[8] Suzuki H. Extracellular Production of Glutathione by Recombinant Microbes. *Foods*. 2023, 14(3):80.

[9] Ma J., Li C., Wang J., Gu J., et al. Genetically engineered *E. coli* Nissle 1917 secreting GLP-1 analog exhibits potential in modulating host metabolism. *Obesity (Silver Spring)*. 2020, 28(2):315-322.

{% 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

* [NMN biosynthesis and detection](#section-1)
  + [Construction of NMN-producing engineered strains](#subsection-2)
  + [Growth activity assay of engineered strains](#subsection-3)
  + [Fermentation conditions](#subsection-4)
  + [NMN quantification](#subsection-5)
  + [Glucose quantification](#subsection-6)
* [GshF synthesis and detection](#section-7)
  + [Construction of GshF-overexpressing engineered strains](#subsection-8)
  + [Enzyme activity assay of GshF](#subsection-9)
  + [Effect of oxygen concentration on GSH production in BL21-GshF](#subsection-10)
  + [Effect of exogenous precursor amino acids on GSH production in BL21-GshF](#subsection-11)
  + [Antioxidant activity assay of GshF by the DPPH method](#subsection-12)
* [PelB-GLP-1 synthesis and detection](#section-13)
  + [Construction of PelB-GLP-1 engineered strains](#subsection-14)
  + [Expression and Western blot analysis of PelB-GLP-1](#subsection-15)
* [Statistical analysis](#section-16)
  + [Related kit instructions](#section-17)

# NMN biosynthesis and detection

## Construction of NMN-producing engineered strains

The coding sequences of *niaP, nadV, baPRS,* and *pnuC* were retrieved from public databases and codon-optimized for *Escherichia coli*. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 assembly standard and compatibility with the pET28a(m) vector. The optimized genes were synthesized (Generalbiol, China) and cloned into the pET28a(m) plasmid using NdeI/XhoI restriction sites, under the control of the T7 promoter. The recombinant plasmid was first transformed into *E. coli* DH5α (D0351, Beyotime) for plasmid amplification and storage, followed by plasmid extraction (Tiangen, China) and transformation into *E. coli* BL21(DE3) (D1009S, Beyotime) for protein expression. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin, verified by colony PCR, and confirmed by Sanger sequencing (Qingke, Beijing). Verified strains were stored in 25% (v/v) glycerol stocks at –80 °C. Routine cultivation was carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin at 37 °C and 200 rpm. In parallel, an empty-vector control strain (BL21-pET28a) was maintained to assess the contribution of the engineered NMN biosynthetic pathway.

## Growth activity assay of engineered strains

To assess growth activity, frozen glycerol stocks of engineered strains and corresponding control strains were thawed and inoculated (1:100, v/v) into pre-warmed selective medium containing the appropriate antibiotic. Cultures were incubated at 37 °C with shaking at 250 rpm for 3–5 h until reaching an OD600 of approximately 0.4–0.6. The bacterial suspensions were then diluted under sterile conditions to normalize the starting OD600 to 0.05–0.20, and 200 μL aliquots were dispensed into transparent 96-well microplates. Blank controls (medium with antibiotic only) and at least three biological replicates were included for each group. Plates were incubated at the target temperature, and OD600 values were recorded at defined time intervals to generate growth curves.

## Fermentation conditions

Frozen glycerol stocks of engineered strains were thawed and inoculated (1:100, v/v) into 5 mL of LB broth supplemented with 100 μg/mL kanamycin in 15 mL culture tubes, and incubated at 37 °C with shaking at 200 rpm for 3–5 h until the culture reached an OD600 of approximately 0.6. Subsequently, 2% (v/v) of the pre-culture was transferred into 30 mL of fresh medium, either LB broth (A507002, Sangon Biotech) or M9 broth (A510881, Sangon Biotech), supplemented with 1% glucose, 1 mM MgSO4 , and 50 μM CaCl2. The cultures were incubated at 37 °C, 200 rpm until reaching an OD600 of 0.2, at which point protein expression was induced with 0.5 mM IPTG. Simultaneously, 0.1% nicotinamide (NAM, A30252, Innochem, China) and 1% glucose (G6500, Innochem, China) were added as precursor and carbon source to promote NMN biosynthesis. Induced cultures were maintained at 37 °C with shaking at 200 rpm and sampled at designated time points for subsequent analysis.

## NMN quantification

### Establishment of NMN standard curve

To enable accurate quantification of NMN, a calibration curve was first established using β-NMN standards (A00974, Innochem, China). Serial dilutions of the standard were subjected to the same fluorescence derivatization procedure as experimental samples. Fluorescence intensity was measured using a microplate reader (FlexStation 3, Molecular Devices, USA) with an excitation wavelength of 382 nm and emission wavelength of 445 nm. A concentration–fluorescence response curve was generated, and linear regression was applied to ensure a correlation coefficient of R2 ≥ 0.98 and to define the validated linear detection range.

### Determination of NMN content

For sample measurement, 1 mL of fermentation broth was centrifuged at 10,000 g for 5 min at 4 °C to separate the supernatant (extracellular fraction) and cell pellet. The pellet was washed with PBS, resuspended, and disrupted on ice using a sonicator (Shanghai Jingxin, China) at 70 W with 1 s on/3 s off cycles for 20 min. Cell lysates were centrifuged at 10,000 g for 30 min at 4 °C, and the resulting supernatant was collected as the intracellular fraction. For derivatization, 69 μL of sample was sequentially mixed with 27.7 μL of 20% acetophenone in DMSO and 27.7 μL of 2 M KOH, incubated on ice for 2 min, followed by the addition of 125 μL of 88% formic acid and incubation at 37 °C for 10 min. The resulting derivatives were analyzed by fluorescence spectroscopy (excitation 382 nm, emission 445 nm), and NMN concentrations were calculated based on the established standard curve.

## Glucose quantification

Glucose concentration in the culture medium was determined using the GOD-POD method (Yeasen, 60408ES60). At designated time points, 1 mL of fermentation broth was collected and centrifuged at 10,000 g for 5 min to remove cells, and the supernatant was used as the assay sample. Samples were diluted appropriately when necessary and mixed with the colorimetric reagents provided in the kit. Absorbance was measured at 505 nm in a 96-well microplate reader, and glucose concentrations were calculated based on a standard calibration curve. The residual glucose levels were then compared with the initial medium concentration to determine glucose consumption during fermentation.

# GshF synthesis and detection

## Construction of GshF-overexpressing engineered strains

The coding sequence of *gshF* (glutathione synthetase fusion) was obtained from GenBank and codon-optimized for *Escherichia coli*. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 standard and compatibility with the multiple cloning site of the pET28a(m) vector. The optimized gene was synthesized commercially (Generalbiol, China) and directionally cloned into the pET28a(m) plasmid via NdeI/XhoI restriction sites under the control of the T7 promoter. The recombinant plasmid was first transformed into *E. coli* DH5α (D0351, Beyotime) for plasmid amplification and storage. Purified plasmids were subsequently transformed into *E. coli* BL21(DE3) (D1009S, Beyotime) for protein expression. Positive colonies were screened on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin and incubated at 37 °C. PCR-verified colonies and further confirmed by Sanger sequencing (Qingke, Beijing), yielding the engineered strain *E. coli* BL21-GshF. Verified recombinant strains were stored in 25% (v/v) glycerol stocks at –80 °C for long-term preservation.

## Enzyme activity assay of GshF

The engineered strain *E. coli* BL21-GshF was inoculated into LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600) of approximately 0.6 was reached. Protein expression was induced with 0.5 mM IPTG at 30 °C for 4 hours. After induction, the cells were harvested by centrifugation at 8000 g, 4 °C for 10 minutes, and washed twice with pre-chilled PBS (pH 7.4). Precisely 1 g of wet cell pellet was resuspended in PBS at a ratio of 1:5 (w/v), followed by sonication on ice (150 W, 1 s on/3 s off cycles, 20 min) to lyse the cells. The lysate was centrifuged at 12,000 g, 4 °C for 15 min, and the resulting supernatant was collected as the crude enzyme extract.

The enzymatic reaction was performed in a 10 mL PBS system containing 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamic acid, 20 mM L-cysteine, and 20 mM glycine. Reactions were incubated at 37 °C for 1 h, then terminated by the addition of 10% (v/v) trichloroacetic acid (TCA). After centrifugation at 12,000 g for 10 min, the supernatant was collected for analysis. The amount of reduced glutathione (GSH) produced was quantified using a commercial detection kit (Solarbio, China). Samples were incubated with DTNB reagent at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentration was calculated based on a standard calibration curve. One unit of enzyme activity (U) was defined as the amount of enzyme required to catalyze the formation of 1 μmol of GSH per minute under the described conditions.

## Effect of oxygen concentration on GSH production in BL21-GshF

The engineered strain *E. coli* BL21-GshF was inoculated into LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600 ) of ~0.6 was reached. The culture was then inoculated at 1% (v/v) into 50 mL LB broth containing 100 μg/mL kanamycin and transferred to an incubator with controlled gas composition. Three oxygen levels were applied: 0% O2 (strict anaerobic conditions), 20% O2 (ambient atmospheric conditions), and 30% O2 (hyperoxic conditions). Cultures were incubated statically at 37 °C for 3 h. At the end of incubation, 1 mL of culture from each group was collected in triplicate, centrifuged at 12,000 g, 4 °C for 10 min, and washed twice with pre-chilled PBS (pH 7.4) to remove residual medium. The washed pellets were resuspended in PBS and lysed by sonication on ice (150 W, 1 s on/3 s off, 20 min). Cell lysates were centrifuged at 12,000 g, 4 °C for 15 min, and the supernatant was collected as the crude enzyme extract. Reduced glutathione (GSH) levels were quantified using a commercial detection kit (DTNB method, Solarbio, China). Extracts were incubated with DTNB at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentrations were calculated from a standard calibration curve, and production levels were compared across different oxygen conditions.

## Effect of exogenous precursor amino acids on GSH production in BL21-GshF

The engineered strain *E. coli* BL21-GshF was inoculated into LB broth containing 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600) of ~0.6 was reached. The seed culture was inoculated at 1% (v/v) into two groups of 50 mL LB broth with kanamycin, with three biological replicates per group to ensure equal starting cell densities. For the experimental group, the medium was supplemented with 20 mM L-glutamic acid, 20 mM L-cysteine, and 20 mM glycine, sterilized by 0.22 μm filtration. The control group received an equal volume of sterile deionized water. Cultures were incubated at 37 °C, 220 rpm for 3 h. At the end of incubation, 1 mL of culture from each replicate was centrifuged at 12,000 g, 4 °C for 10 min, and cell pellets were washed twice with pre-chilled PBS (pH 7.4). Washed cells were resuspended in PBS and disrupted by sonication on ice. The lysates were centrifuged, and the supernatant was collected as the crude enzyme extract. GSH content was quantified using a reduced glutathione detection kit (DTNB method, Solarbio, China). Extracts were incubated with DTNB at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentrations were determined using a standard calibration curve, and the results were compared between the experimental and control groups.

## Antioxidant activity assay of GshF by the DPPH method

Frozen glycerol stocks of *E. coli* BL21-GshF were inoculated (100 μL) into 5 mL LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C, 200 rpm for 12 h. The pre-culture was then transferred at a 1:100 inoculation ratio into 100 mL LB broth with kanamycin and grown until reaching an OD600 of ~0.6. Cells were harvested by centrifugation at 8000 g, 4 °C for 10 min, washed twice with pre-chilled PBS (0.1 mol/L, pH 7.4), and resuspended in 10 mL PBS. Cell suspensions were disrupted on ice by sonication (150 W, 3 s on/5 s off cycles, 20 min), followed by centrifugation at 12,000 g, 4 °C for 20 min. The resulting supernatant was filtered through a 0.22 μm membrane to obtain the crude enzyme extract.

For antioxidant activity determination, a 0.1 mmol/L DPPH working solution was prepared in absolute ethanol and stored at 4 °C in the dark. A GSH standard curve was established using serial dilutions (0, 20, 40, 60, 80, 100 μmol/L) in PBS (0.1 mol/L). For the assay, 50 μL of sample supernatant or GSH standard was mixed with 50 μL of DPPH solution in a 96-well microplate and incubated at 25 °C in the dark for 30 min. Absorbance was measured at 517 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). The radical scavenging activity was expressed as DPPH clearance percentage, calculated using the formula:

![Antioxidant activity assay of GshF by the DPPH method Diagram](https://static.igem.wiki/teams/5985/experiments/img1.webp)

# PelB-GLP-1 synthesis and detection

## Construction of PelB-GLP-1 engineered strains

The coding sequence of the PelB-GLP-1 fusion protein was retrieved from public databases and codon-optimized for *Escherichia coli*. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 standard and compatibility with the pET28a(m) vector. The optimized gene was synthesized commercially (Generalbiol, China) and cloned into the pET28a(m) plasmid using NdeI/XhoI restriction sites, under the control of the T7 promoter for expression. The recombinant plasmid was first transformed into *E. coli* DH5α (D0351, Beyotime) for plasmid amplification and storage, and subsequently extracted and introduced into *E. coli* BL21(DE3) (D1009S, Beyotime) for protein expression. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin and incubated at 37 °C. PCR-verified colonies and further confirmed by Sanger sequencing (Qingke, Beijing). Verified recombinant strains were stored in 25% (v/v) glycerol stocks at –80 °C for long-term preservation.

## Expression and Western blot analysis of PelB-GLP-1

For expression, *E. coli* BL21(DE3) carrying the PelB-GLP-1 construct was first inoculated into 3 mL LB broth containing 100 μg/mL kanamycin and cultured overnight at 37 °C, 220 rpm. The pre-culture was then expanded into 50 mL LB + kanamycin and incubated at 37 °C until reaching an OD600 of 0.5–0.8, followed by induction with IPTG at a final concentration of 0.2–0.5 mM for 4 h at 37 °C. Cells were harvested by centrifugation at 10,000 g, 4 °C for 10 min, and the supernatant was collected. Protein samples were mixed with 5× reducing sample buffer and denatured at 95–100 °C for 5–10 min, then cooled on ice. Proteins were separated by 15% SDS-PAGE at 120 V and transferred to PVDF membranes under wet transfer conditions (100 V, 75 min, 4 °C). Membranes were blocked with 5% skim milk in TBST at room temperature for 1 h, then incubated overnight at 4 °C with anti-His antibody (mouse, Beyotime AH367, 1:1000). After washing three times with TBST (10 min each), membranes were incubated with HRP-conjugated goat anti-mouse IgG (Beyotime A0216, 1:1000) at room temperature for 1 h, followed by three additional washes. Finally, membranes were incubated with ECL reagents (A: B = 1:1) for 1–2 min and imaged using a chemiluminescence detection system according to the manufacturer’s protocol.

# Statistical analysis

All experiments were carried out with at least three independent repeats to ensure reliability. Data were processed and visualized using GraphPad Prism. Results are shown as mean ± SD, with individual data points displayed whenever possible. Statistical comparisons between groups were performed using one-way ANOVA with Tukey’s test for multiple groups, or Student’s t-test for two-group comparisons. A p-value less than 0.05 was considered statistically significant.

# Related kit instructions

Plasmid Extraction Kit (DP103)

Plasmid Extraction Kit (DP103).pdf

0.9MB

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Reduced Glutathione (GSH) Content Detection Kit Instruction Manual

Reduced Glutathione (GSH) Content Detection Kit Instruction Manual.pdf

0.3MB

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Glucose Content Detection Kit (GOD-POD, Microplate Method)

Glucose Content Detection Kit (GOD-POD, Microplate Method).pdf

0.1MB

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{% 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: NMN Biosynthesis Module](#section-2)
    - [Effect of NAM on *E. coli* BL21 Growth](#subsection-3)
    - [Establishment of an NMN Standard Curve](#subsection-4)
    - [Establishing Baseline NMN Production with NadV and NiaP (Version 1.0)](#subsection-5)
    - [Optimal NAM Concentration Enhances NMN Production](#subsection-6)
    - [Enhancing NMN Production with BaPRS (Version 2.0)](#subsection-7)
    - [Assessing Growth Activity of NMN-Engineered Strains](#subsection-8)
    - [M9 Medium Enhances NMN Production Compared to LB](#subsection-9)
    - [PnuC Enhancing NMN Export and Increases Accumulation (Version 3.0)](#subsection-10)
    - [Linking NMN Production to Glucose Utilization and Growth Activity](#subsection-11)
    - [Determining the Optimal Temperature for NMN Production](#subsection-12)
  + [System 2: GSH Biosynthesis Module](#section-13)
    - [Enhancing GSH Production by GshF Overexpression](#subsection-14)
    - [Effect of GshF Overexpression on Bacterial Growth](#subsection-15)
    - [GshF Enzyme Activity Assay](#subsection-16)
    - [Effect of Precursor Amino Acids on GSH Production](#subsection-17)
    - [Effect of Oxygen on GSH Production](#subsection-18)
  + [System 3: GLP-1 Biosynthesis Module](#section-19)
    - [Objective](#subsection-20)
    - [Method](#subsection-21)
    - [Result](#subsection-22)
    - [Conclusion](#subsection-23)
  + [Reference](#section-24)

# Abstract:

Our project set out to explore whether engineered *E. coli* could serve as a living platform against aging by integrating three functional modules. In System 1, we established NMN biosynthesis with *NiaP* and *NadV*, and enhanced production through *BaPRS* and *PnuC*, confirming efficient intracellular synthesis and extracellular accumulation. In System 2, we enabled one-step glutathione (GSH) biosynthesis by overexpressing *GshF*, which significantly increased GSH yield and improved antioxidant capacity. In System 3, we validated GLP-1 secretion using a PelB fusion design, confirmed by PCR and Western blot. Collectively, these results demonstrate that engineered bacteria can stably produce NMN, accumulate GSH, and secrete GLP-1, providing a modular synthetic biology framework for probiotic-based anti-aging strategies.

# System 1: NMN Biosynthesis Module

## Effect of NAM on *E. coli* BL21 Growth

#### Objective

To investigate the effect of various concentrations of the substrate nicotinamide (NAM) on the growth of engineered *Escherichia coli* BL21 (DE3), *E. coli* BL21, we measured cell density over time under five NAM conditions. This experiment aimed to determine whether increasing NAM concentrations promotes or inhibits biomass accumulation, providing a reference for optimizing fermentation conditions in NMN production.

#### Method

To assess the effect of nicotinamide (NAM) on host cell growth, *E. coli* BL21 was cultured in LB broth supplemented with kanamycin. NAM was added to final concentrations of 0.1%, 1%, and 5% (w/v), with a parallel control group lacking NAM. Cultures were incubated at 37 °C with shaking at 200 rpm. At predetermined time points, samples were collected and cell density was measured at OD600 using a microplate reader (Figure 1). All conditions were performed in triplicate, and the average OD600 values were used to compare growth performance across different NAM concentrations.

![Effect of NAM on <em>E. coli</em> BL21 Growth Diagram](https://static.igem.wiki/teams/5985/results/img1.webp)

Figure 1. Workflow for Bacterial Growth Measurement.

#### Result

As shown in Figure 2, supplementation with 0.1% NAM had no significant effect on BL21 growth compared with the control. At 1% NAM, growth was still observed but reached a slightly lower density. In contrast, 5% NAM supplementation markedly inhibited bacterial proliferation, with cultures showing a much slower increase in OD600. These results demonstrate that *E. coli* can tolerate low to moderate NAM concentrations, while high concentrations strongly suppress cell growth, likely due to osmotic stress and interference with NAD+ metabolism.

![Effect of NAM on <em>E. coli</em> BL21 Growth Diagram](https://static.igem.wiki/teams/5985/results/img2.webp)

Figure 2. Effect of Different NAM Concentrations on the Growth of Engineered *E. coli* BL21.

#### Conclusion

These results confirmed that while low NAM concentrations (≤1%) did not strongly affect growth, high NAM supplementation (5%) markedly inhibited proliferation, most likely due to osmotic stress and interference with NAD+ metabolism. This highlighted the need to carefully balance precursor supply in subsequent experiments, guiding us to test whether NMN biosynthesis could be enabled by transporter and enzyme introduction.

## Establishment of an NMN Standard Curve

### Objective

To establish a reliable standard curve for NMN quantification, enabling the conversion of fluorescence signals into NMN concentrations for accurate determination of intra- and extracellular NMN levels.

### Method

As shown in Figure 3, β-NMN standards of known purity were prepared in a series of gradient concentrations (e.g., 0, 1, 2, 5, 10, 20 μM). Each concentration was subjected to fluorescence derivatization following the experimental protocol, and fluorescence intensity was measured using a microplate reader with excitation at 382 nm and emission at 445 nm. Each point was tested in triplicate, and the average intensity was plotted against the standard concentrations. A linear regression analysis was performed to generate the calibration curve, recording the slope, intercept, and coefficient of determination (R2).

![Establishment of an NMN Standard Curve Diagram](https://static.igem.wiki/teams/5985/results/img3.webp)

Figure 3. Workflow for NMN Detection.

### Result

As shown in Figure 4, fluorescence intensity exhibited a strong linear correlation with NMN concentration across the tested range. The regression equation was Y = 1.154·X -25.58, and the calibration curve showed excellent linearity (R2 > 0.98), confirming that the method can reliably convert fluorescence signals into NMN concentrations.

![Establishment of an NMN Standard Curve Diagram](https://static.igem.wiki/teams/5985/results/img4.webp)

Figure 4. The standard curve of NMN.

### Conclusion

A highly linear NMN standard curve was successfully established, enabling accurate quantification of NMN in both intracellular and extracellular fractions. This calibration ensures the precision and reliability of subsequent NMN measurement experiments.

## Establishing Baseline NMN Production with NadV and NiaP (Version 1.0)

### Objective

Previous studies have demonstrated that NadV is a key determinant for NMN biosynthesis in *E. coli* [1,2].To establish a baseline NMN biosynthesis system, we introduced two key components into *E. coli* BL21: the nicotinamide transporter *NiaP*, which facilitates efficient uptake of extracellular nicotinamide (NAM), and the nicotinamide phosphoribosyltransferase *NadV*, which catalyzes the conversion of NAM into NMN. By combining substrate uptake with catalytic conversion, this “Version 1.0” design aimed to determine whether *E. coli* could be endowed with the capacity to synthesize NMN, thereby validating the feasibility of constructing a microbial NMN biosynthetic pathway.

### Method

The engineered strains were constructed using standard molecular cloning procedures (Figure 5). The coding sequences of *NiaP* and *NadV* were obtained from public databases and codon-optimized for expression in *E. coli*. Restriction enzyme recognition sites incompatible with RFC#10 assembly (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed during sequence design to ensure cloning compatibility. The optimized fragments were synthesized commercially and sequentially assembled into the pET28a backbone under the control of the T7 promoter, ribosome binding site B0034, and terminator B0015. Recombinant plasmids were initially transformed into *E. coli* DH5α for amplification. Positive colonies were screened on LB agar with 100 μg/mL kanamycin and verified by colony PCR and Sanger sequencing (Figure 6). The validated constructs were then introduced into *E. coli* BL21, generating BL21-NiaP and BL21-NiaP-NadV strains, while an empty-vector control (BL21-pET28a, namely BL21) was maintained for comparison.

![Establishing Baseline NMN Production with NadV and NiaP (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/results/img5.webp)

Figure 5. Workflow for Engineering Strain Construction.

For the NAM precursor assay, BL21-NiaP-NadV and control strains were cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. NAM was added at different concentrations (0.05%, 0.1%, 0.5%, and 1%) together with 1% glucose. Cultures were incubated at 37 °C with shaking for 8 h. After harvesting and lysis, NMN levels were quantified via acetophenone-based fluorescence derivatization (Ex 382 nm / Em 445 nm), with concentrations determined using the previously established standard curve.

![Establishing Baseline NMN Production with NadV and NiaP (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/results/img6.webp)

Figure 6. Construction and validation of engineered strain BL21-NiaP-NadV.

### Result

As shown in Figure 7, the wild-type BL21 strain produced no detectable NMN across all measured time points, confirming that *E. coli* lacks an inherent NMN biosynthetic capability. In contrast, the engineered strain BL21-NiaP-NadV exhibited a clear and time-dependent increase in NMN accumulation following IPTG induction. NMN levels began to rise steadily at 2 h post-induction, reached a marked increase at 4 h, and peaked at approximately 6 h before plateauing. This demonstrates that while *NiaP* alone was insufficient, the combined expression of *NiaP* and *NadV* endowed *E. coli* with the capacity to synthesize measurable levels of NMN.

![Establishing Baseline NMN Production with NadV and NiaP (Version 1.0) Diagram](https://static.igem.wiki/teams/5985/results/img7.webp)

Figure 7. NMN production in BL21-NiaP-NadV.

### Conclusion

The results confirmed that *E. coli* BL21 alone cannot synthesize NMN, but equipping the strain with *NiaP* and *NadV* successfully enabled intracellular NMN production. While *NiaP* facilitated NAM uptake, *NadV* proved essential for catalytic conversion. This established a functional baseline (Version 1.0) and provided the foundation to investigate whether precursor limitation could be alleviated to further boost NMN yield.

## Optimal NAM Concentration Enhances NMN Production

### Objective

To investigate how different concentrations of nicotinamide (NAM) influence NMN production in the engineered *E. coli* BL21-NiaP-NadV strain.

### Method

BL21-NiaP-NadV was cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. To test precursor dependency, NAM was added at various concentrations (e.g., 0.05%, 0.1%, 0.5%, and 1%), together with 1% glucose as a carbon source. Cultures were incubated at 37 °C with shaking for 8 h. Samples were collected, lysed, and subjected to fluorescence-based derivatization. NMN levels were calculated according to the previously established standard curve.

### Result

As shown in Figure 8, NMN production was strongly dependent on NAM concentration. At low supplementation (0.05%), NMN levels remained limited, only slightly above background. Increasing NAM to 0.1% led to a marked improvement, with NMN concentrations rising significantly compared to the low-dose group. Further increase to 0.5% NAM resulted in the highest NMN yield, indicating that sufficient precursor supply enhanced biosynthesis efficiency. However, raising NAM to 1% did not further boost NMN accumulation and instead showed a plateau or slight decline, suggesting that excessive NAM may impose metabolic stress or feedback inhibition on the pathway.

![Optimal NAM Concentration Enhances NMN Production Diagram](https://static.igem.wiki/teams/5985/results/img8.webp)

Figure 8. Effect of NAM concentration on NMN production in BL21-NiaP-NadV.

### Conclusion

We observed that NMN production was strongly dependent on NAM concentration, with 0.1% NAM supporting the highest yield. Excessive NAM not only failed to increase production but also suppressed biosynthesis, consistent with its inhibitory effect on growth. These findings indicated that precursor availability must be finely tuned, prompting us to explore whether pathway engineering could overcome PRPP limitation.

## Enhancing NMN Production with BaPRS (Version 2.0)

### Objective

Building on our previous findings—that high concentrations of NAM inhibit *E. coli* growth, NMN can be reliably quantified by fluorescence-based derivatization, NadV is the key determinant for NMN biosynthesis, and excessive NAM reduces production efficiency—we next aimed to address the limitation of precursor supply in the pathway. Since phosphoribosyl pyrophosphate (PRPP) availability is a major bottleneck, we introduced *Bacillus mycoides* phosphoribosylpyrophosphate synthetase (*BaPRS*) into BL21-NiaP-NadV to evaluate whether enhanced PRPP generation could increase NMN yield and improve pathway efficiency [3].

### Methods

The coding sequences of *NiaP*, *NadV*, and *BaPRS* were obtained from public databases and codon-optimized for expression in *Escherichia coli*. During sequence design, incompatible restriction enzyme recognition sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 assembly standard and compatibility with the multiple cloning site of the pET28a vector. The optimized fragments were synthesized commercially and sequentially cloned into the pET28a plasmid under the control of the T7 promoter, with ribosome binding site B0034 and terminator B0015. Correct integration of *BaPRS* was verified by PCR amplification (expected 951 bp band) and Sanger sequencing (Figure 9). The validated plasmid was then transformed into *E. coli* BL21 to generate the engineered strain BL21-NiaP-NadV-BaPRS for subsequent functional analysis.

![Enhancing NMN Production with BaPRS (Version 2.0) Diagram](https://static.igem.wiki/teams/5985/results/img9.webp)

Figure 9. Construction and validation of BL21-NiaP-NadV-BaPRS.

The engineered strain BL21-NiaP-NadV-BaPRS was cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, with 0.1% NAM and glucose provided as substrates. Cultures were incubated at 37 °C with shaking at 200 rpm. Samples were collected at different time points post-induction (0 h, 2 h, 4 h, 6 h, 8 h). Cells were harvested, lysed, and analyzed by fluorescence-based derivatization. NMN concentrations were quantified using the previously established standard curve.

### Result

As shown in Figure 10, the BL21-NiaP-NadV-BaPRS strain produced markedly higher NMN levels compared with the baseline BL21-NiaP-NadV strain. NMN accumulation began to rise at 2 h, showing a steady upward trend through the 4 h and 6 h time points. By 8 h, the engineered strain reached its maximum NMN concentration, which was significantly greater than that of Version 1.0 without *BaPRS*. This result indicates that *BaPRS* effectively boosted NMN biosynthesis by alleviating precursor limitation from PRPP.

![Enhancing NMN Production with BaPRS (Version 2.0) Diagram](https://static.igem.wiki/teams/5985/results/img10.webp)

Figure 10. NMN production in BL21-NiaP-NadV-BaPRS over time.

### Conclusion

The introduction of *BaPRS* significantly enhanced NMN accumulation, confirming that phosphoribosyl pyrophosphate (PRPP) availability was a major bottleneck in the initial pathway. By boosting intracellular PRPP levels, *BaPRS* increased the catalytic efficiency of *NadV*. This Version 2.0 design thus demonstrated the value of reinforcing precursor supply, paving the way to test whether transport engineering could further improve NMN accumulation.

## Assessing Growth Activity of NMN-Engineered Strains

### Objective

To evaluate whether the introduction of NMN biosynthetic modules affects host cell viability, we compared the growth performance of control BL21 and engineered strains harboring different pathway constructs (Version 1.0: NiaP-NadV; Version 2.0: NiaP-NadV-BaPRS). This experiment aimed to confirm that NMN production does not severely compromise bacterial growth under induction conditions.

### Method

Control BL21, BL21-NiaP-NadV, and BL21-NiaP-NadV-BaPRS strains were cultured in LB broth supplemented with kanamycin, 0.1% NAM, and 1% glucose. When cultures reached OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. Cell density was subsequently measured at OD600 at defined time intervals using a microplate reader throughout cultivation. Growth curves were then plotted to compare proliferation dynamics across strains under identical conditions.

### Result

As shown in Figure 11, all three strains—control BL21, BL21-NiaP-NadV, and BL21-NiaP-NadV-BaPRS—entered exponential growth after IPTG induction and reached comparable stationary phases within 10–12 h. The control BL21 exhibited the fastest early growth, while BL21-NiaP-NadV showed a slightly delayed lag phase, likely due to the metabolic burden of expressing additional genes. Notably, the BaPRS-containing strain (BL21-NiaP-NadV-BaPRS) maintained a growth trajectory similar to the control, suggesting that the reinforcement of PRPP supply did not impose additional metabolic stress. These results indicate that NMN pathway engineering, including the introduction of BaPRS, preserved host viability and supported stable proliferation under NAM- and glucose-supplemented conditions.

![Assessing Growth Activity of NMN-Engineered Strains<em>                   </em> Diagram](https://static.igem.wiki/teams/5985/results/img11.webp)

Figure 11. Growth curves of control and NMN-engineered BL21 strains.

### Conclusion

The results confirm that engineered strains carrying NMN biosynthetic modules remain viable and capable of sustained growth under induction conditions. Although a slight reduction in growth was observed, particularly in the BaPRS-enhanced strain, the overall impact on cellular activity was minimal. This demonstrates that NMN pathway engineering is compatible with normal host physiology, ensuring strain stability for further optimization.

## M9 Medium Enhances NMN Production Compared to LB

### Objective:

To investigate the impact of medium composition on NMN biosynthesis, we compared NMN production in engineered *E. coli* strains cultured in rich medium (LB) versus defined medium (M9). Since medium components may influence precursor supply and enzyme activity, this experiment aimed to identify culture conditions that maximize NMN yield.

### Method

Engineered strains BL21-NiaP-NadV and BL21-NiaP-NadV-BaPRS were cultured in LB broth or M9 minimal medium (Sangon Biotech, A510881). Both media were supplemented with 1% glucose and 0.1% NAM. For M9, additional supplements included 1 mM MgSO4 and 50 μM CaCl2. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, and cultures were incubated for 12 h at 37 °C with shaking. After induction, samples were harvested, and intracellular NMN levels were quantified using fluorescence-based derivatization and the previously established standard curve.

### Results

As shown in Figure 12, strains cultured in M9 medium accumulated substantially higher NMN compared to those grown in LB medium under identical conditions. Both BL21-NiaP-NadV and BL21-NiaP-NadV-BaPRS exhibited this trend, with the BaPRS-enhanced strain in M9 reaching the highest NMN yield after 12 h induction. The data indicate that minimal medium with appropriate supplementation provided a more favorable environment for NMN biosynthesis, while LB supported lower levels of accumulation.

![M9 Medium Enhances NMN Production Compared to LB Diagram](https://static.igem.wiki/teams/5985/results/img12.webp)

Figure 12. Effect of culture medium (LB vs. M9) on NMN production in engineered strains.

### Conclusion

These results demonstrate that medium composition strongly influences NMN production efficiency. The defined M9 medium enhanced NMN accumulation compared with LB, particularly in the BaPRS-containing strain. This suggests that precursor balance and cofactors in the medium may play critical roles in supporting NMN biosynthesis. Potential explanations include increased phosphate availability in M9 to sustain PRPP-dependent reactions, stabilization of membrane transport and secretion by Ca2+, and Mg2+ serving as a cofactor for BaPRS or NadV.

## PnuC Enhancing NMN Export and Increases Accumulation (Version 3.0)

### Objective

Building upon Version 2.0, which alleviated precursor limitations by introducing BaPRS, we next sought to address the challenge of NMN export. While intracellular accumulation can be enhanced by boosting precursor supply, the efficiency of NMN production is ultimately constrained if export into the culture medium is limited. To overcome this, we introduced the NMN transporter PnuC, which has been reported to mediate nucleoside mononucleotide export [4].

### Method

The coding sequence of the NMN transporter *PnuC* was obtained from public databases, codon-optimized for expression in *Escherichia coli*, and designed to remove restriction enzyme sites incompatible with RFC#10 assembly. The optimized fragment was synthesized commercially and sequentially cloned into the previously constructed pET28a backbone containing *NiaP*, *NadV*, and *BaPRS*, under the control of the T7 promoter with ribosome binding site B0034 and terminator B0015. Recombinant plasmids were first amplified in *E. coli* DH5α, verified by colony PCR and Sanger sequencing, and subsequently transformed into *E.* *coli* BL21 to generate the final engineered strain BL21-NiaP-NadV-BaPRS-PnuC (Figure 13). Verified strains were preserved in 25% glycerol at –80 °C for long-term storage.

![PnuC Enhancing NMN Export and Increases Accumulation (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/results/img13.webp)

Figure 13. Construction and validation of BL21-NiaP-NadV-BaPRS-PnuC.

Engineered strains BL21-NiaP-NadV-BaPRS (Version 2.0) and BL21-NiaP-NadV-BaPRS-PnuC (Version 3.0) were cultured in LB broth supplemented with kanamycin, 0.1% NAM, and 1% glucose. At OD600 ≈ 0.2, IPTG was added to a final concentration of 0.5 mM to induce expression. Cultures were incubated at 37 °C with shaking for 12 h. After induction, both intracellular and extracellular fractions were collected separately by centrifugation. NMN concentrations were quantified using fluorescence-based derivatization and the established standard curve.

### Result

As shown in Figure 14, the BL21-NiaP-NadV-BaPRS-PnuC strain exhibited a significant increase in NMN accumulation compared with both the Version 1.0 (NiaP-NadV) and Version 2.0 (NiaP-NadV-BaPRS) strains. While earlier constructs produced detectable NMN, the addition of *PnuC* resulted in notably higher levels, indicating that export efficiency was a critical factor in maximizing overall NMN yield.

![PnuC Enhancing NMN Export and Increases Accumulation (Version 3.0) Diagram](https://static.igem.wiki/teams/5985/results/img14.webp)

Figure 14.Effect of PnuC introduction on NMN accumulation in engineered strains.

### Conclusion

Introducing *PnuC* markedly increased extracellular NMN accumulation, highlighting the importance of transporter engineering for metabolite secretion. By coupling *NiaP*-mediated import, *NadV*- and *BaPRS*-mediated synthesis, and *PnuC*-mediated export, we successfully constructed an efficient three-step system (Version 3.0). This completed the core NMN production module and established a strong basis for linking NMN biosynthesis with host physiology in subsequent studies.

## Linking NMN Production to Glucose Utilization and Growth Activity

### Objective

To investigate the relationship between NMN biosynthesis, glucose consumption, and bacterial activity, we monitored the growth performance of the Version 3.0 strain BL21-NiaP-NadV-BaPRS-PnuC alongside measurements of NMN accumulation and glucose utilization. This experiment aimed to assess whether NMN production imposes a metabolic burden on the host and how carbon source availability influences yield.

### Method

The engineered strain BL21-NiaP-NadV-BaPRS-PnuC was cultivated in LB broth containing kanamycin, 0.1% NAM, and 1% glucose. At mid-log phase (OD600 ≈ 0.2), protein expression was induced with 0.5 mM IPTG, and cultures were incubated at 37 °C with shaking for 12 h. At defined intervals, aliquots were collected and subjected to three parallel analyses: (1) OD600 measurement to monitor cell growth, (2) quantification of residual glucose concentration using the GOD-POD enzymatic assay, and (3) determination of NMN accumulation via fluorescence-based derivatization. The overall experimental workflow is illustrated in Figure 15.

![Linking NMN Production to Glucose Utilization and Growth Activity Diagram](https://static.igem.wiki/teams/5985/results/img15.webp)

Figure 15. Workflow for Glucose Detection.

### Result

As shown in Figure 16, glucose consumption was closely correlated with both cell growth and NMN accumulation in BL21-NiaP-NadV-BaPRS-PnuC. During the exponential growth phase, residual glucose levels decreased sharply, while NMN concentration increased correspondingly, indicating that active metabolism supported efficient biosynthesis. By the stationary phase, glucose was largely depleted, cell growth plateaued, and NMN production reached its maximum level. This strong linkage highlights the dependence of NMN biosynthesis on glucose utilization.

![Linking NMN Production to Glucose Utilization and Growth Activity Diagram](https://static.igem.wiki/teams/5985/results/img16.webp)

Figure 16. Growth, glucose consumption, and NMN accumulation of BL21-NiaP-NadV-BaPRS-PnuC.

### Conclusion

These results demonstrate that glucose availability directly influences NMN production efficiency, with rapid glucose consumption during exponential growth driving high NMN yields. Once glucose became limiting, both growth and NMN synthesis declined, suggesting that carbon source supplementation or controlled feeding strategies could further optimize production. This finding provides a metabolic basis for future bioprocess design aimed at scaling NMN biosynthesis.

## Determining the Optimal Temperature for NMN Production

### Objective

To identify the optimal temperature for NMN production in the engineered strain BL21-NiaP-NadV-BaPRS-PnuC, we compared product yields at different cultivation temperatures. Although *E. coli* typically grows best at 37 °C, lower induction temperatures are sometimes used to improve the expression of heterologous proteins. This experiment aimed to determine whether temperature modulation could enhance NMN biosynthesis.

### Method

BL21-NiaP-NadV-BaPRS-PnuC was cultured in LB medium supplemented with kanamycin (100 μg/mL), 1% glucose, and 0.1% NAM. When cultures reached OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. To specifically evaluate the effect of temperature, all culture conditions (medium composition, inducer concentration, shaking speed, and induction time) were kept constant, while incubation was carried out at different temperatures (25 °C, 30 °C, and 37 °C) with shaking at 200 rpm. After 12 h of cultivation, cells were harvested, lysed, and NMN concentrations were quantified using fluorescence-based derivatization and the established standard curve.

### Result

As shown in Figure 17, NMN production was strongly influenced by cultivation temperature. At 25 °C, NMN accumulation increased slowly and plateaued at a relatively low level by 12 h, indicating suboptimal enzyme activity under cooler conditions. In contrast, cultures grown at 30 °C and 37 °C exhibited significantly higher NMN yields, with both conditions showing a steady upward trend throughout the cultivation period. This result suggests that while the engineered strain can produce NMN across a broad temperature range, moderate-temperature induction at 30 °C provides the most favorable balance between cell growth and protein expression, thereby representing the optimal condition for large-scale production.

![Determining the Optimal Temperature for NMN Production Diagram](https://static.igem.wiki/teams/5985/results/img17.webp)

Figure 17. Effect of cultivation temperature on NMN production in BL21-NiaP-NadV-BaPRS-PnuC.

### Conclusion

Temperature optimization experiments confirmed that 37 °C remains the most suitable condition for NMN production in BL21-NiaP-NadV-BaPRS-PnuC. Unlike some recombinant protein expression systems where low-temperature induction enhances yield, NMN biosynthesis in this pathway is favored at the standard physiological growth temperature of *E. coli*.

# System 2: GSH Biosynthesis Module

## Enhancing GSH Production by GshF Overexpression

### Objective

Building on our success in establishing a functional NMN biosynthetic pathway, we next sought to enhance the cellular antioxidant capacity as a complementary anti-aging strategy. Glutathione (GSH), a tripeptide composed of glutamate, cysteine, and glycine, is one of the most important intracellular antioxidants, playing a key role in redox homeostasis and defense against oxidative stress. While GSH biosynthesis in most organisms requires two sequential enzymes—γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS)—the bifunctional enzyme GshF combines both activities within a single polypeptide, enabling one-step GSH synthesis [5]. In this study, we aimed to evaluate whether overexpression of *GshF* in *E. coli* BL21 could significantly increase intracellular GSH accumulation compared with the control strain.

### Method

The coding sequence of *GshF*, a bifunctional enzyme involved in glutathione biosynthesis, was obtained from public databases, codon-optimized for expression in *Escherichia coli*, and redesigned to remove restriction sites incompatible with RFC#10 assembly (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI). The optimized fragment was synthesized commercially and cloned into the pET28a vector under the T7 promoter with ribosome binding site B0034 and terminator B0015. The recombinant plasmid (pET28a-*GshF*) was first transformed into *E. coli* DH5α for plasmid amplification, with positive clones verified by kanamycin selection, colony PCR (expected 2,253 bp band), and Sanger sequencing (Figure 18). Verified plasmids were subsequently introduced into *E. coli* BL21 to generate the engineered strain BL21-*GshF*, which was preserved in 25% (v/v) glycerol at –80 °C for long-term storage.

![Enhancing GSH Production by GshF Overexpression Diagram](https://static.igem.wiki/teams/5985/results/img18.webp)

Figure 18. Construction and validation of BL21-GshF.

For GSH production assays, BL21-GshF and control BL21 were cultured in LB broth containing kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, and cultures were incubated at 30 °C with shaking at 200 rpm. After 8 h, cells were harvested, lysed, and intracellular GSH levels were quantified using the DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) colorimetric method (Figure 19).

![Enhancing GSH Production by GshF Overexpression Diagram](https://static.igem.wiki/teams/5985/results/img19.webp)

Figure 19. Workflow for GSH Detection.

### Result

As shown in Figure 20, BL21-*GshF* exhibited significantly elevated intracellular GSH levels compared with the control BL21 strain. Following IPTG induction, engineered cells displayed a marked increase in GSH accumulation within 8 h, whereas the control strain maintained only basal levels. These findings demonstrate that overexpression of *GshF* successfully enhanced glutathione biosynthesis in *E. coli*.

![Enhancing GSH Production by GshF Overexpression Diagram](https://static.igem.wiki/teams/5985/results/img20.webp)

Figure 20. Intracellular GSH levels in control BL21 and BL21-GshF strains.

### Conclusion

Overexpressing *GshF* in BL21 enabled one-step GSH biosynthesis and significantly increased intracellular GSH levels compared with the control strain. This confirmed the feasibility of using a bifunctional enzyme to simplify the pathway. Building on this foundation, we next examined whether *GshF* overexpression affected cell growth and metabolic balance.

## Effect of GshF Overexpression on Bacterial Growth

### Objective

To evaluate whether overexpression of the fusion enzyme GshF, in addition to enhancing glutathione (GSH) biosynthesis, exerts any impact on host cell growth, we compared the growth curves of BL21-GshF with the wild-type BL21. Since elevated antioxidant capacity could alleviate intracellular oxidative stress, it was hypothesized that GshF expression might promote bacterial proliferation.

### Method

BL21-GshF and wild-type BL21 were cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Growth was monitored by measuring OD600 at defined time intervals over a 12 h cultivation period using a microplate reader. Growth curves were plotted to assess differences in proliferation between the engineered and control strains.

### Result

As shown in Figure 21, both strains exhibited typical bacterial growth curves with lag, exponential, and stationary phases. However, BL21-GshF consistently displayed higher OD600 values compared to the wild-type, particularly during the exponential phase. After 12 h, the engineered strain reached an OD600 of approximately 1.45, whereas the control strain reached only about 1.05, indicating a growth advantage of ~38%. The enhanced proliferation suggests that elevated GSH levels provided by GshF expression may mitigate oxidative stress and improve cellular fitness under cultivation conditions.

![Effect of GshF Overexpression on Bacterial Growth Diagram](https://static.igem.wiki/teams/5985/results/img21.webp)

Figure 21. Growth curves of wild-type BL21 and BL21-GshF.

### Conclusion

The growth curve analysis showed that *GshF*-overexpressing strains exhibited no significant growth defects compared with the control, demonstrating that enhanced GSH production did not compromise cell viability. This result indicated that the engineered strains could sustain both growth and production, encouraging us to proceed with characterizing enzyme activity.

## GshF Enzyme Activity Assay

### Objective

To confirm that the overexpressed GshF fusion enzyme retained its catalytic activity in *E. coli*, we directly assayed the intracellular enzymatic function by quantifying glutathione (GSH) production. The aim was to validate that the observed increase in GSH levels was indeed attributable to GshF activity rather than background metabolic processes.

### Method

BL21-GshF and wild-type BL21 strains were cultured in LB broth and induced with 0.5 mM IPTG at OD600 ≈ 0.2. After 12 h of cultivation, cells were harvested and lysed by sonication. Enzyme activity was determined indirectly by measuring intracellular GSH content using the DTNB assay, where reduced glutathione reacts with DTNB to generate a yellow-colored product measurable at 412 nm. GSH concentration was quantified against a standard curve, and enzymatic activity was expressed in terms of μmol GSH produced per mg total protein per min.

### Result

As shown in Figure 22, BL21-GshF exhibited significantly higher GSH levels compared with the wild-type control. The engineered strain achieved an enzymatic activity of approximately 3.2-fold greater than the baseline, confirming that the recombinant GshF fusion enzyme was both correctly expressed and functionally active in *E. coli*. This elevated activity directly correlates with the enhanced intracellular GSH accumulation observed in previous experiments.

![GshF Enzyme Activity Assay Diagram](https://static.igem.wiki/teams/5985/results/img22.webp)

Figure 22. Enzymatic activity assay confirming functional GshF expression in *E. coli*.

### Conclusion

The activity assay confirmed that the recombinant *GshF* enzyme retained strong catalytic activity, directly contributing to elevated GSH biosynthesis. This provided a mechanistic basis for the increased GSH observed in vivo. With enzymatic activity validated, we next explored how precursor amino acid supplementation influenced production efficiency.

## Effect of Precursor Amino Acids on GSH Production

### Objective

Glutathione (GSH) biosynthesis requires three precursor amino acids: glutamate, cysteine, and glycine. To determine whether external supplementation of these substrates could enhance GSH production in *E. coli* BL21-GshF, we evaluated intracellular GSH levels under different amino acid supplementation conditions.

### Method

BL21-GshF was cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Experimental groups were provided with additional precursor amino acids (glutamate, cysteine, glycine, or a combination of all three), while the control group was cultured without supplementation. After 12 h of cultivation, cells were harvested, lysed, and the GSH concentration was quantified using the DTNB assay.

### Result

As shown in Figure 23, amino acid supplementation enhanced GSH accumulation compared with the unsupplemented control. Among the single-supplement groups, cysteine exerted the most significant effect, followed by glutamate and glycine. Notably, supplementation with all three precursors simultaneously resulted in the highest GSH yield, approximately 2.8-fold greater than the control. This indicates that precursor availability directly influences metabolic flux toward GSH biosynthesis.

![Effect of Precursor Amino Acids on GSH Production Diagram](https://static.igem.wiki/teams/5985/results/img23.webp)

Figure 23. Effect of precursor amino acid supplementation on GSH production in BL21-GshF.

### Conclusion

Supplementation with cysteine significantly enhanced GSH production, while providing all three precursors—glutamate, cysteine, and glycine—further maximized yield. This result is consistent with previous reports that optimizing precursor availability can markedly improve glutathione production in *E. coli* [6]. Together, these findings underscore the necessity of a balanced precursor supply for efficient GSH biosynthesis. Building on this insight, we next examined how environmental factors, particularly oxygen availability, influence production.

## Effect of Oxygen on GSH Production

### Objective

Glutathione (GSH) plays a central role in mitigating oxidative stress, and its biosynthesis is closely linked to the redox environment of the cell. Oxygen availability influences both intracellular oxidative pressure and enzyme activity. To assess the relationship between oxygen levels and GSH production, we cultured BL21-GshF under different aeration conditions, aiming to determine whether oxidative stress could serve as a driving force for enhanced GSH biosynthesis.

### Method

BL21-GshF was cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Cultures were divided into three groups: anaerobic, aerobic (normal shaking at 200 rpm), and high-aeration (vigorous shaking at 250 rpm with increased flask-to-medium ratio). After 12 h of cultivation, cells were harvested, lysed, and intracellular GSH levels were quantified using the DTNB assay.

### Result

As shown in Figure 24, oxygen levels significantly influenced GSH production. Under anaerobic conditions, GSH accumulation was minimal, reflecting the absence of oxidative stress stimuli and reduced metabolic demand for antioxidant protection. Under normal aerobic conditions, GSH production increased substantially, consistent with the role of GSH in counteracting reactive oxygen species generated during respiration. Strikingly, the high-aeration group produced the highest GSH yield, approximately 3.5-fold higher than anaerobic cultures. This result suggests that increased oxidative stress triggered by elevated oxygen availability stimulated GshF-mediated glutathione synthesis.

![Effect of Oxygen on GSH Production Diagram](https://static.igem.wiki/teams/5985/results/img24.webp)

Figure 24. Effect of oxygen availability on GSH production in BL21-GshF.

### Conclusion

Higher oxygen availability significantly increased GSH accumulation, reflecting the close link between redox conditions and glutathione metabolism. These results emphasized that both genetic and environmental factors jointly shape GSH biosynthesis. To assess the functional relevance of enhanced GSH production, we next evaluated the antioxidant capacity of the engineered strains.

### Antioxidant Activity Assay (DPPH Radical Scavenging)

### Objective

While intracellular measurements confirmed that GshF overexpression increased glutathione (GSH) production, it was also important to validate whether this enhancement translated into improved antioxidant capacity. To address this, we assessed the free radical scavenging ability of BL21-GshF compared with the wild-type strain using the DPPH assay, a standard method for evaluating antioxidant activity.

### Method

BL21-GshF and wild-type BL21 were cultured in LB medium with kanamycin, induced with 0.5 mM IPTG, and harvested after 12 h of growth. Cells were lysed, and crude extracts were prepared. Antioxidant capacity was measured using the DPPH free radical scavenging assay, in which extracts were incubated with DPPH solution, and the decrease in absorbance at 517 nm was recorded. Radical scavenging activity was calculated relative to the control reaction without cell extract.

### Result

As shown in Figure 25, BL21-GshF exhibited significantly stronger radical scavenging activity than wild-type BL21. The engineered strain achieved approximately 2.6-fold higher DPPH scavenging efficiency, reflecting its elevated GSH content. This result confirmed that the increased glutathione pool directly improved the antioxidant potential of the engineered cells.

![Effect of Oxygen on GSH Production Diagram](https://static.igem.wiki/teams/5985/results/img25.webp)

Figure 25. Antioxidant capacity of BL21-GshF measured by DPPH radical scavenging assay.

### Conclusion

The DPPH assay demonstrated that GshF overexpression not only boosted intracellular glutathione production but also translated into enhanced antioxidant functionality. This validates the biological significance of the GSH system, positioning engineered *E. coli* as a potential chassis for applications in oxidative stress resistance and anti-aging biotechnology.

# System 3: GLP-1 Biosynthesis Module

## Objective

Building on the successful establishment of NMN and GSH biosynthesis modules, we next sought to construct a GLP-1 biosynthesis module to broaden the functional scope of our engineered system. Glucagon-like peptide-1 (GLP-1) is a key regulatory peptide with important roles in glucose metabolism and anti-aging pathways. To achieve microbial production, we engineered *E. coli* BL21 to express GLP-1 fused with the PelB signal peptide, enabling extracellular secretion. This experiment aimed to validate the feasibility of GLP-1 expression and secretion in bacteria, thereby laying the foundation for future functional and therapeutic applications.

## Method

To investigate the feasibility of GLP-1 secretion, the GLP-1 coding sequence was fused with the PelB signal peptide, following a design strategy inspired by the iGEM24\_Squirrel-CHN team. The fusion gene was codon-optimized for expression in *Escherichia coli*, with incompatible restriction sites removed to ensure RFC#10 assembly compatibility, and subsequently synthesized commercially. The optimized fragment was cloned into the pET28a backbone under the control of a T7 promoter, ribosome binding site B0034, and terminator B0015, forming a complete expression cassette. The recombinant plasmid was propagated in *E. coli* DH5α and validated by PCR amplification and agarose gel electrophoresis, which produced a distinct 162 bp band; sequencing further confirmed construct integrity. The verified plasmid was then transformed into *E. coli* BL21 to generate the engineered strain BL21-PelB-GLP-1 (Figure 26).

![Method Diagram](https://static.igem.wiki/teams/5985/results/img26.webp)

Figure 26. Construction and validation of the PelB-GLP-1 expression cassette.

To confirm the expression of GLP-1 in the engineered strain, BL21-PelB-GLP-1 cultures were induced with IPTG, and protein samples were collected at the designated time points. Cells were harvested by centrifugation, lysed, and the soluble protein fraction was separated by SDS-PAGE. Proteins were subsequently transferred onto a PVDF membrane, probed with an anti-GLP-1 primary antibody, and detected using an HRP-conjugated secondary antibody. Signal visualization was performed with enhanced chemiluminescence (ECL), following the workflow illustrated in Figure 27.

![Method Diagram](https://static.igem.wiki/teams/5985/results/img27.webp)

Figure 27. Workflow for Western Blot Analysis.

## Result

As shown in Figure 28, a distinct immunoreactive band of under 15 kDa was detected in protein extracts from BL21-PelB-GLP-1 after IPTG induction. This band size corresponds to the expected molecular weight of the PelB-GLP-1 fusion protein, confirming its successful expression in the engineered strain. Together, these results validate that the PelB signal peptide enabled detectable GLP-1 expression at the protein level.

![Result Diagram](https://static.igem.wiki/teams/5985/results/img28.webp)

Figure 28. Western Blot validation of PelB-GLP-1 expression.

## Conclusion

The GLP-1 secretion module was successfully established through the construction and validation of the PelB-GLP-1 expression cassette. PCR and sequencing confirmed the correct integration of the fusion gene, while Western blot analysis revealed a distinct immunoreactive band at the expected molecular weight (under 15 kDa), verifying that the engineered strain BL21-PelB-GLP-1 expressed the target protein. The presence of the PelB signal peptide indicates that the system holds the potential for extracellular secretion, providing a practical route for GLP-1 delivery.

# Reference

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[3] Black W, Wei Y, Yang S. Metabolic engineering of *Escherichia coli* for optimized nicotinamide mononucleotide biosynthesisJJJ. *Metabolic Engineering*, 2020, 61: 80-90. DOI:10.1016/j.ymben.2020.06.006.

[4] Luo X, Sun J, Zhou J, et al. Biosynthesis of nicotinamide mononucleotide: current strategies and challengesJJJ. *Fermentation*, 2023, 9(7): 594. DOI:10.3390/fermentation9070594.

[5] Stout J, Schuller D, Meury M, et al. Molecular characterization of GshF, a bifunctional enzyme in bacterial glutathione biosynthesisJJJ. *Journal of Bacteriology*, 2011, 193(6): 1461-1470. DOI:10.1128/JB.01175-10.

[6] Zhang J, Xu D, Feng X, et al. Systematic manipulation of glutathione metabolism in *Escherichia coli* for improved productionJJJ. *Microbial Cell Factories*, 2016, 15: 38. DOI:10.1186/s12934-016-0435-3.

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