iGEM 2025 — Team bsdfz-china

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

![](https://static.igem.wiki/teams/6029/common/content-title.webp)

* [System 1-Sakuranetin production system（06/25–07/29）- 93 pages](#section-1)
  + [📊 Weekly Gantt Chart](#subsection-2)
  + [📅 Daily Experiment Schedule](#subsection-3)
* [System 2-LPC Production System（07/29–08/13）- 16 pages](#section-4)
  + [📊 Weekly Gantt Chart](#subsection-5)
  + [📅 Daily Experiment Schedule](#subsection-6)
* [System 3-Safety Suicide System（08/21–08/31）- 19 pages](#section-7)
  + [📊 Weekly Gantt Chart](#subsection-8)
  + [📅 Daily Experiment Schedule](#subsection-9)

# System 1-Sakuranetin production system（06/25–07/29）- 93 pages

From June 25 to July 29, 2025, we completed the construction and optimization of the (2S)-sakuranetin production system in *E. coli*. First, BL21-OMT was built by introducing the F7-OMT gene, which enabled sakuranetin synthesis with an optimal substrate concentration of 150 mg/L naringenin, producing ~49 mg/L sakuranetin. Supplementation with methionine further increased yield by ~55%, confirming the role of SAM as a methyl donor. In the second-generation system, YdaO was co-expressed with OMT, doubling sakuranetin production (~36 mg/L vs. ~19 mg/L) by improving ATP regulation. In the third-generation system, CysE was added to enhance SAM biosynthesis, leading to the highest yield (~45 mg/L), a 2.4-fold improvement compared to the first generation. Meanwhile, the Nar system was successfully constructed by expressing TAL/4CL and CHS/CHI operons in BL21, enabling de novo naringenin biosynthesis, with growth and glucose consumption verified in 48 h fermentation. Finally, all data were consolidated and visualized with GraphPad Prism, confirming the stepwise optimization strategy from substrate supplementation to metabolic pathway engineering.

## 📊 Weekly Gantt Chart

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/6029/notebook/img1.webp)

## 📅 Daily Experiment Schedule

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 2025-06-25 | F7-OMT Gene Reception & LB Media Preparation | Received synthetic gene (F7-OMT); prepared LB agar plates and LB broth with Amp; sterilized tools | Amp added at ~55℃; plates stored inverted at 4℃; gene solution aliquoted to avoid degradation |
| 2025-06-26 | PCR Amplification of F7-OMT + Double Digestion (pSB1A3) | PCR of F7-OMT; gel extraction of ~1.1 kb band; pSB1A3 digested with XbaI/SpeI | Minimized UV exposure; ensured full gel dissolution; stored DNA in aliquots |
| 2025-06-27 | T4 Ligation of F7-OMT & Transformation into DH5α | Ligated F7-OMT into pSB1A3; transformed into DH5α; plated on LB Amp | Negative control plate included; colonies expected next day |
| 2025-06-28 | Screening DH5α Transformants (Colony PCR + Miniprep) | Picked 10 colonies; colony PCR ; plasmid miniprep of positive clones | colonies positive; plasmids prepared for sequencing |
| 2025-06-29 | Sequencing & BL21(DE3) Transformation | Verified sequence of F7-OMT plasmids; transformed into BL21(DE3); glycerol stocks prepared | 3 correct clones selected; both DH5α & BL21 versions preserved |
| 2025-06-30 | BL21-OMT Pre-culture & Fermentation Start | Revived BL21-OMT; inoculated TB medium with 150 mg/L naringenin; started fermentation | Substrate pre-dissolved in methanol; sampling after 24 h planned |
| 2025-07-01 | HPLC Detection of Sakuranetin | HPLC analysis of BL21 vs BL21-OMT cultures; sakuranetin detected at ~24.5 min | BL21 negligible (~0.65 mg/L), BL21-OMT ~49.5 mg/L |
| 2025-07-02 | Effect of Substrate Concentrations | Tested 100/150/300 mg/L naringenin in BL21-OMT; HPLC analysis | Optimal at 150 mg/L (~50 mg/L sakuranetin); high concentration (300 mg/L) inhibited yield |
| 2025-07-03 | Effect of Methionine Addition | Compared BL21-OMT ± 1 g/L methionine; fermentation + HPLC | Methionine increased yield by ~55% (26.56 vs 17.16 mg/L) |
| 2025-07-04 | PCR YdaO + Double Digestion (pSB1A3) | Attempted PCR of YdaO and digestion of pSB1A3-OMT | Failed — no clear PCR band; possible primer/template/enzyme issues |
| 2025-07-05 | Repeat PCR YdaO + Double Digestion (pSB1A3) | Optimized PCR (higher template & annealing temp); successful YdaO band; pSB1A3 digested | Insert and vector ready for ligation |
| 2025-07-06 | T4 Ligation of OMT-YdaO + DH5α Transformation | Ligated YdaO with OMT backbone; transformed into DH5α; plated | Colonies grew well; negative control clean |
| 2025-07-07 | Colony PCR Screening of OMT-YdaO | Screened 12 colonies; 8 positive for YdaO insert | Positive clones cultured overnight for plasmid extraction |
| 2025-07-08 | Plasmid Miniprep + Sequencing of OMT-YdaO | Miniprep of 8 clones; NanoDrop 80–150 ng/μL; sequencing submitted | Sequencing primers VF2/VR + internal YdaO used |
| 2025-07-09 | BL21(DE3) Transformation with OMT-YdaO | Transformed sequencing-verified OMT-YdaO plasmid into BL21(DE3); glycerol stocks prepared | Colonies robust; BL21 transformants ready for fermentation |
| 2025-07-10 | Comparative Fermentation (BL21-OMT vs BL21-OMT-YdaO) | Fermentation with 150 mg/L naringenin; HPLC after 12 h | BL21-OMT ~18.96 mg/L; BL21-OMT-YdaO ~36.10 mg/L (~2× higher) |
| 2025-07-11 | PCR Amplification of CysE + Double Digestion (OMT-YdaO) | Amplified CysE; digested OMT-YdaO plasmid with XbaI/SpeI | Prepared DNA fragments for next ligation |
| 2025-07-12 | T4 Ligation of CysE with OMT-YdaO Backbone + DH5α Transformation | Ligated gel-purified CysE insert into digested OMT-YdaO plasmid backbone; transformed into DH5α; plated on LB Amp | Negative control plate clean; colonies expected after overnight growth |
| 2025-07-13 | Colony PCR Screening of OMT-YdaO-CysE Transformants | Picked 12 colonies, performed colony PCR with primers across OMT–CysE junction; expected ~2.2 kb band | 9/12 colonies positive; positive colonies cultured overnight for plasmid extraction |
| 2025-07-14 | Plasmid Miniprep of Positive OMT-YdaO-CysE Clones | Miniprep of overnight cultures using Tiangen kit; DNA quantified with NanoDrop | Yield 90–150 ng/μL; purity A260/280 = 1.8–1.95; plasmids stored at -20℃ |
| 2025-07-15 | Sequencing Verification of OMT-YdaO-CysE Plasmids | Sent plasmids for Sanger sequencing with VF2/VR + internal primers | Sequencing confirmed correct construct; no mutations observed |
| 2025-07-16 | BL21(DE3) Transformation with Verified OMT-YdaO-CysE Plasmid | Transformed confirmed plasmid into BL21(DE3) competent cells; plated and obtained colonies | Colonies robust; glycerol stocks prepared at -80℃; BL21-OMT-YdaO-CysE strain established |
| 2025-07-17 | Comparative Fermentation Test of OMT, OMT-YdaO, OMT-YdaO-CysE | Fermentation with 150 mg/L naringenin for 12 h; HPLC analysis of sakuranetin production | BL21-OMT ~18.9 mg/L; BL21-OMT-YdaO ~31.1 mg/L; BL21-OMT-YdaO-CysE ~45.2 mg/L |
| 2025-07-18 | Extended Fermentation (24 h) of OMT, OMT-YdaO, OMT-YdaO-CysE | Repeated fermentation extended to 24 h; samples collected for HPLC | Experiment completed but HPLC data lost due to computer malfunction during export |
| 2025-07-19 | (Reserved for Repeat of 24 h Fermentation) | Planned repeat of 0718 fermentation due to data loss; same setup with 150 mg/L naringenin | Experiment postponed, scheduled for later |
| 2025-07-20 | PCR Amplification of TAL/4CL and CHS/CHI Operons | Amplified TAL/4CL and CHS/CHI from synthetic templates; purified by gel extraction | Gradient PCR used (58–62℃); best at 60℃; DNA stored at -20℃ |
| 2025-07-21 | Restriction Digestion of pSB1A3 + Ligation of TAL/4CL & CHS/CHI | Digested pSB1A3 with XbaI/SpeI; ligated TAL/4CL and CHS/CHI operons into backbone; transformed DH5α | Dozens of colonies observed; vector-only control plate had no colonies |
| 2025-07-22 | Colony PCR Screening of DH5α-Nar Transformants | Picked 12 colonies, PCR with primers spanning TAL→CHI region; | 8/12 colonies positive; positive clones grown overnight in LB Amp |
| 2025-07-23 | Plasmid Miniprep + Sequencing of pSB1A3-Nar | Miniprep from overnight cultures; sequencing with VF2/VR and internal primers | Sequencing confirmed correct TAL/4CL + CHS/CHI assembly |
| 2025-07-24 | Transformation of Verified pSB1A3-Nar into BL21(DE3) | Transformed sequencing-confirmed plasmid into BL21(DE3); plated on LB Amp | Multiple colonies obtained; glycerol stocks prepared; BL21-Nar strain constructed |
| 2025-07-25 | BL21-Nar Fermentation Setup + 0 h & 12 h Sampling | Fermentation in M9 medium (0.4% glucose, 3 mM tyrosine, 1 mM MgSO₄, 50 μM CaCl₂, 340 mg/L thiamine); OD600 and glucose measured | OD600 rose from ~0.004→0.12; glucose dropped from ~3.99→3.58 g/L |
| 2025-07-26 | BL21-Nar 24 h & 36 h Sampling | Continued fermentation; OD600 and glucose measured at 24 h & 36 h | OD600 rose to ~0.30; glucose declined to ~1.94 g/L; consistent with active metabolism |
| 2025-07-27 | BL21-Nar 48 h Final Sampling | Collected OD600 and glucose data at 48 h; consolidated 0–48 h dataset | Final OD600 ~0.42; glucose ~1.55 g/L; fermentation profile complete |
| 2025-07-28～2025-07-29 | Data Consolidation + GraphPad Analysis (Growth/Glucose) | Compiled OD600 & glucose data; generated growth curve and glucose consumption curve in GraphPad | Saved plots in .png and .tif; raw GraphPad files archived |

system 1 notebook.pdf

5.8MB

[Download](https://static.igem.wiki/teams/6029/notebook/system-1.pdf)

# System 2-LPC Production System（07/29–08/13）- 16 pages

In System 2, we successfully constructed an engineered *E. coli* BL21(DE3) strain overexpressing phospholipase A2 (PLA2). The PLA2 gene was codon-optimized for *E. coli* and cloned into the pET28a(m) vector, followed by verification in DH5α and transfer into BL21(DE3). Expression conditions were optimized using IPTG induction under different concentrations and temperatures. Western blot analysis confirmed robust PLA2 expression after repeating the experiment. Functional assays demonstrated that the engineered strain effectively degraded phosphatidylcholine (PC), producing lysophosphatidylcholine (LPC), as quantified by ELISA. Compared with wild-type BL21, the BL21-PLA2 strain showed significantly enhanced LPC accumulation, confirming successful enzyme activity. This system establishes a reliable platform for downstream studies of phospholipid metabolism and engineered LPC production.

## 📊 Weekly Gantt Chart

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/6029/notebook/img2.webp)

## 📅 Daily Experiment Schedule

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 07/29 | Gene Reception & Preparation | Receive synthetic PLA2 gene; prepare LB+Kana broth and plates; sterilize glassware. | Kanamycin added <55 °C; plates stored inverted at 4 °C. |
| 07/30 | PCR Verification & Double Digestion | PCR amplify PLA2; NdeI/XhoI digestion of insert and pET28a(m); gel check and extraction. | Minimize UV exposure; DNA stored at −20 °C for ligation. |
| 07/31 | T4 Ligation & DH5α Transformation | Ligate PLA2 insert with pET28a(m); transform into DH5α; recover in SOC; plate on LB+Kana. | Vector-only control included; colonies incubated overnight. |
| 08/01 | Colony PCR Screening | Pick colonies; run PCR with PLA2 primers; check ~800 bp band; inoculate positives. | Multiple positives kept for reliability. |
| 08/04 | Transformation into BL21 (DE3) | Transform sequencing-verified plasmid into BL21 (DE3); plate on LB+Kana. | No-DNA control included. |
| 08/05 | Pre-culture of BL21-PLA2 | Inoculate BL21-PLA2 and controls into 5 mL LB+Kana; grow overnight. | Clones clearly labeled. |
| 08/06–07 | Expression & SDS-PAGE | Induce with IPTG (0.1–0.5 mM); 37 °C vs 25–30 °C; sample at 0/4/8 h; lysis + SDS-PAGE. | Collected both supernatant and pellet for WB. |
| 08/08 | Western Blot (Attempt 1) | Transfer proteins; primary anti-PLA2 (1:1000); HRP secondary; ECL detection. | No visible band detected. |
| 08/09 | Western Blot (Repeat) | Increased loading & extended transfer; detected clear PLA2 band (30~42 kDa). | Controls negative; confirmed PLA2 expression. |
| 08/10 | PLA2 Activity Assay (ELISA) | Fermentation with IPTG + PC; collect supernatant; filter; dilute; ELISA for LPC. | Standard curve prepared; triplicates performed. |
| 08/12 | Statistical Analysis & Visualization | Compile data (BL21 vs BL21-PLA2); t-test; plot bar + dot plots in GraphPad Prism. | R² ≥0.98 for curve; figures exported in .png/.tif formats. |

system 2 notebook.pdf

0.6MB

[Download](https://static.igem.wiki/teams/6029/notebook/system-2-notebook.pdf)

# System 3-Safety Suicide System（08/21–08/31）- 19 pages

System 3 focused on developing a dual-layer safety suicide system in *E. coli* to minimize biosafety risks. First, the arabinose-inducible promoter pBAD was validated using mRFP as a reporter, showing dose-dependent expression, and then coupled with T4 holin and T4 lysozyme to form the pBAD–T4 lysis module. Upon arabinose induction, engineered strains underwent rapid lysis within 8–20 h, while control strains grew normally. In parallel, the cold-inducible promoter pCspA was tested with mRFP, demonstrating strong expression at 16 ℃ and minimal background at 37 ℃. Replacing mRFP with T4 lysis yielded the pCspA–T4 lysis system, which effectively restricted bacterial growth under low-temperature conditions. Together, these results confirm that System 3 combines artificial clearance (via arabinose induction) with environment-triggered lysis (via cold shock), establishing a reliable and complementary dual-safeguard mechanism for engineered strains.

## 📊 Weekly Gantt Chart

![📊 Weekly Gantt Chart  Diagram](https://static.igem.wiki/teams/6029/notebook/img3.webp)

## 📅 Daily Experiment Schedule

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 08/21 | Construct pBAD-mRFP reporter | Synthesize/verify pBAD(BBa\_K808000)+B0034+mRFP; clone into pSB1A3 via XbaI/SpeI; transform DH5α on LB+Amp. | Prepare L-arabinose stock solutions at different concentrations. |
| 08/22 | Colony PCR & overnight culture | Pick 8–12 colonies, colony PCR across pBAD–mRFP; inoculate positives into LB+Amp overnight. | Keep master plate; record clone IDs. |
| 08/23 | Sequencing & prep for testing | Miniprep positive clones, send sequencing; prepare 0/0.05%/0.2%/0.5% L-arabinose; set sampling times (0/2/4/8 h). | Early test can be done with one confirmed positive. |
| 08/24 | pBAD-mRFP induction test | 1:100 inoculation into LB+Amp; arabinose gradient; 37 ℃, 180 rpm; measure fluorescence (Ex584/Em607) and OD600, calculate Flu/OD. | Include non-induced blank; three biological replicates. |
| 08/25 | Construct pBAD-T4 lysis | Place T4 holin+T4 lysozyme downstream of pBAD-B0034; clone into pSB1A3 via XbaI/SpeI; transform DH5α. | Verify reading frame and terminator; include empty vector control. |
| 08/26 | Screening & pre-culture | Colony PCR for T4H and T4L fragments; inoculate positives into LB+Amp overnight. | Prepare for lysis assay next day. |
| 08/27 | pBAD-T4 lysis test | Grow at 37 ℃, add 0.5 mM L-arabinose at 4 h; measure OD600 at 0/2/4/8/20 h. | Run DH5α control in parallel; record onset of lysis. |
| 08/28 | Construct pCspA-mRFP reporter | Synthesize/verify pCspA(BBa\_K4987003)+mRFP; clone into pSB1A3 via XbaI/SpeI; transform DH5α. | Reserve incubators at 16/25/30/37 ℃. |
| 08/29 | Screening & cold induction test | Colony PCR positives; 1:100 inoculation LB+Amp; incubate at 16/25/30/37 ℃ for 12 h; measure fluorescence and OD600, calculate Flu/OD. | Focus on strong induction at 16 ℃ and low background at 37 ℃. |
| 08/30 | Construct pCspA-T4 lysis & test | Clone T4 lysis downstream of pCspA; transform DH5α; test at 16 ℃, 180 rpm, measure OD600 at intervals. | Expect significant growth inhibition/lysis at low temperature. |
| 08/31 | Data integration & visualization | Summarize pBAD (dose-time response) and pCspA (temperature response) data; generate Flu/OD and OD vs time plots; finalize dual-suicide system report. | Export .png/.tif figures and raw tables; complete Notebook text. |

system 3 notebook.pdf

0.8MB

[Download](https://static.igem.wiki/teams/6029/notebook/system-3-notebook.pdf)

{% 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 %}

![](https://static.igem.wiki/teams/6029/common/content-title.webp)

* [Abstract](#section-1)
  + [Cycle 1-1 BL21-OMT](#section-2)
    - [Design](#subsection-3)
    - [Build](#subsection-4)
    - [Test](#subsection-5)
    - [Learn](#subsection-6)
  + [Cycle 1-2 BL21-OMT-YdaO](#section-7)
    - [Design](#subsection-8)
    - [Build](#subsection-9)
    - [Test](#subsection-10)
    - [Learn](#subsection-11)
  + [Cycle 1-3 BL21-OMT-YdaO-CysE](#section-12)
    - [Design](#subsection-13)
    - [Build](#subsection-14)
    - [Test](#subsection-15)
    - [Learn](#subsection-16)
  + [Cycle 1-4 BL21-Nar](#section-17)
    - [Design](#subsection-18)
    - [Build](#subsection-19)
    - [Test](#subsection-20)
    - [Learn](#subsection-21)
  + [Cycle 2 BL21-PLA2](#section-22)
    - [Design](#subsection-23)
    - [Build](#subsection-24)
    - [Test](#subsection-25)
    - [Learn](#subsection-26)
  + [Cycle 3-1 DH5α–pBAD–mRFP](#section-27)
    - [Design](#subsection-28)
    - [Build](#subsection-29)
    - [Test](#subsection-30)
    - [Learn](#subsection-31)
  + [Cycle 3-2 DH5α–pBAD–T4 lysis](#section-32)
    - [Design](#subsection-33)
    - [Build](#subsection-34)
    - [Test](#subsection-35)
    - [Learn](#subsection-36)
  + [Cycle 3-3 DH5α–pCspA–mRFP](#section-37)
    - [Design](#subsection-38)
    - [Build](#subsection-39)
    - [Test](#subsection-40)
    - [Learn](#subsection-41)
  + [Cycle 3-4 DH5α–pCspA–T4 lysis](#section-42)
    - [Design](#subsection-43)
    - [Build](#subsection-44)
  + [Test](#section-45)
    - [Learn](#subsection-46)
  + [Reference](#section-47)

# Abstract

We achieved measurable gains through three engineering cycles focused on efficacy and biosafety. Cycle 1 (Sakuranetin production): In BL21-OMT (24 h), sakuranetin increased from 0.65 ± 0.05 mg/L (control) to 49.46 ± 9.53 mg/L. The optimal substrate was 150 mg/L naringenin (50.21 ± 2.67 mg/L), while 300 mg/L caused inhibition (26.47 ± 5.45 mg/L). Adding 1 g/L methionine raised titers from 17.16 ± 1.67 to 26.56 ± 0.80 mg/L (+55%), indicating SAM limitation. Co-expression of YdaO (12 h) improved yield from 18.96 ± 3.06 to 36.10 ± 2.19 mg/L (~+90%); adding CysE further increased it to 45.21 ± 3.75 mg/L (12 h), demonstrating that strengthening energy/SAM supply relieves the methyl-donor bottleneck. To eliminate exogenous substrate dependence, BL21-Nar produced naringenin endogenously to 0.4214 ± 0.0474 g/L (48 h), while glucose dropped from 39.88 ± 1.70 to 15.48 ± 2.68 g/L, enabling future one-pot coupling. Cycle 2 (LPC production): In BL21-PLA2, Western blot showed a clear ~30–42 kDa band; functionally, LPC rose from 2.43 ± 1.28 μg/mL (control) to 74.23 ± 15.91 μg/mL , establishing a second intervention route. Cycle 3 (dual-layer biosafety): pBAD-mRFP confirmed low leakiness and dose/time-dependent induction; pBAD-T4 reduced OD600 to 0.19 ± 0.16 at 20 h post-induction; pCspA-mRFP was strongly ON at 16 ℃ and near-baseline at 37 ℃; pCspA-T4 markedly suppressed growth at 16 ℃ (8 h: control 0.83 ± 0.06 vs engineered 0.34 ± 0.08) while remaining silent at 37 ℃. Together, we delivered dual functional modules (sakuranetin + LPC) on a chassis equipped with manual (pBAD-T4) + environmental (pCspA-T4) containment. Next, we will couple BL21-Nar with the OMT module to achieve “endogenous supply + in situ conversion” in a single strain, further increasing titer and translational potential.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/imgex-new.avif)

# Cycle 1-1 BL21-OMT

## Design

Sakuranetin, a dihydroflavonoid compound extracted from plants, has been reported to exert potential beneficial effects against Alzheimer’s disease. To enable *E. coli* to autonomously synthesize sakuranetin, we designed a metabolic circuit that uses (2S)-naringenin as the substrate. In this pathway, F7-OMT (flavonoid 7-O-methyltransferase) catalyzes the methylation reaction, with S-adenosylmethionine (SAM) serving as the methyl donor, thereby converting (2S)-naringenin into (2S)-sakuranetin.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img1.webp)

Figure: Conversion of naringenin to sakuranetin catalyzed by F7-OMT using SAM as the methyl donor.

## Build

The F7-OMT gene (BBa\_259M31WV, Generalbiol, China) was synthesized and codon-optimized for efficient expression in *E. coli*. To comply with the RFC#10 standard, restriction sites EcoRI, XbaI, SpeI, and PstI were removed. The gene was placed under the control of the constitutive promoter J23100 and ribosome binding site B0034, and cloned into the pSB1A3 vector using XbaI and SpeI restriction sites. The recombinant plasmid was first transformed into *E. coli* DH5α for propagation. Positive clones were screened on LB agar plates (1.5% agar) supplemented with 100 μg/mL ampicillin and verified by sequencing (Qingke, Beijing), yielding the strain DH5α-OMT. Plasmids were then extracted (DP103, Tiangen) and subsequently transformed into *E. coli* BL21 (DE3) to generate the engineered strain BL21-OMT.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img2.webp)

Figure: Construction of BL21-OMT. (A) The plasmid map of pSB1A3-OMT. (B) Agarose gel electrophoresis of F7-OMT (1056 bp). (C) The gene circuit of BL21-OMT.

For long-term preservation, strains were stored at –80 ℃ in 25% (v/v) glycerol. For routine cultivation, BL21-OMT was grown at 37 ℃ and 150 rpm in LB broth (G3102, Servicebio, China) containing 100 μg/mL ampicillin.

## Test

### HPLC analysis of naringenin and sakuranetin

To evaluate the catalytic efficiency of the constructed strain, BL21-OMT was cultured in TB medium supplemented with 150 mg/L naringenin at 25 ℃ for 24 h. Following fermentation, 1 mL of culture broth was mixed with 1 mL methanol, centrifuged at 12,000 × g for 5 min, and the supernatant was filtered through a 0.22 μm nylon membrane. Samples were analyzed by HPLC using an Agilent C18 column (4.6 × 250 mm, 5 μm) with detection at 290 nm. The mobile phases were water with 0.1% TFA (A) and acetonitrile with 0.1% TFA (B), run at 1 mL/min with the gradient: 0–10 min, 10% → 60% B; 10–20 min, 40% → 80% B; 20–25 min, 80% → 10% B. Standard curves for naringenin and sakuranetin were established using authentic references to enable quantitative analysis.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img3.webp)

Figure：High performance liquid chromatography（HPLC）to detect naringenin and sakuranetin

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img4.webp)

Figure: HPLC chromatograms of naringenin and sakuranetin

HPLC analysis of BL21-OMT fermentation broth revealed two distinct and well-resolved peaks: naringenin at ~12.4 min and sakuranetin at ~24.5 min, matching the retention times of standards. The peaks were sharp and symmetrical with stable baselines, demonstrating excellent resolution and reproducibility. The established calibration curves showed strong linearity, allowing reliable conversion of peak areas into concentrations. This validated method enabled accurate quantification of substrate consumption and product accumulation, providing a robust tool for assessing sakuranetin production efficiency in engineered strains.

### F7-OMT promotes the production of sakuranetin

After establishing a reliable HPLC detection method, we compared sakuranetin production between the engineered strain BL21-OMT and the control strain BL21. Both strains were cultured in TB medium supplemented with 150 mg/L naringenin and incubated at 25 ℃ for 24 h. At the end of fermentation, culture samples were processed by methanol extraction, centrifugation, and 0.22 μm filtration, followed by quantitative analysis of sakuranetin using HPLC.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img5.webp)

Figure：Sakuranetin production in BL21 and BL21-OMT strains after 24 h fermentation

The control strain BL21 produced only a trace amount of sakuranetin (0.65 ± 0.05 mg/L). In contrast, the engineered strain BL21-OMT exhibited a markedly higher yield of 49.46 ± 9.53 mg/L, representing an approximately 76-fold increase. These results demonstrate that expression of F7-OMT effectively promoted the conversion of naringenin into sakuranetin.

### Effect of naringenin concentration on sakuranetin production

To investigate the effect of substrate concentration on sakuranetin production, BL21-OMT was cultured in TB medium supplemented with 100 mg/L, 150 mg/L, or 300 mg/L naringenin at 25 ℃ for 24 h. Samples were processed and analyzed by HPLC as described above to quantify sakuranetin yields under each condition.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img6.webp)

Figure: Sakuranetin production of BL21-OMT under different naringenin concentrations

Statistical analysis confirmed that substrate concentration had a significant effect on production (ANOVA, F = 21.09, p = 0.0019 < 0.05)：

* 100 mg/L naringenin: sakuranetin yield was 39.19 ± 4.84 mg/L;
* 150 mg/L naringenin: yield reached 50.21 ± 2.67 mg/L, the highest among all conditions;
* 300 mg/L naringenin: yield decreased significantly to 26.47 ± 5.45 mg/L.

The results indicate that 150 mg/L naringenin is the optimal substrate concentration for achieving the highest sakuranetin yield.

### Methionine supplementation promotes sakuranetin production

To evaluate the impact of methionine supplementation on sakuranetin production, BL21-OMT was cultured with 150 mg/L naringenin as substrate under two conditions: without methionine and with 1 g/L methionine supplementation. Cultures were incubated at 25 ℃ for 24 h, and sakuranetin titers were quantified by HPLC following the established protocol.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img7.webp)

Figure：Effect of 1 g/L methionine supplementation on sakuranetin production in BL21-OMT

Under conditions without methionine supplementation, BL21-OMT produced only 17.16 ± 1.67 mg/L of sakuranetin; with 1 g/L methionine, production increased significantly to 26.56 ± 0.80 mg/L, representing an approximate 55% improvement. This demonstrates that exogenous methionine supplementation effectively promotes sakuranetin synthesis, likely by enhancing the intracellular availability of S-adenosylmethionine (SAM), the essential methyl donor for OMT-catalyzed reactions. Thus, SAM supply is identified as a key limiting factor, providing a clear direction for subsequent engineering efforts to optimize the Met–SAM cycle.

## Learn

In the BL21-OMT cycle, multiple tests provided key insights into the system’s performance. First, we established a reliable HPLC method capable of distinguishing and quantifying both the substrate naringenin (12.4 min) and the product sakuranetin (24.5 min), ensuring accurate yield analysis. Next, we confirmed the effect of F7-OMT: while the control strain produced only 0.65 ± 0.05 mg/L sakuranetin, BL21-OMT achieved a significantly higher yield of 49.46 ± 9.53 mg/L, validating the feasibility of the designed pathway. We then examined substrate concentration and found that 150 mg/L naringenin gave the highest production (50.21 ± 2.67 mg/L), whereas excessive substrate at 300 mg/L reduced production to 26.47 ± 5.45 mg/L, indicating substrate inhibition. Finally, methionine supplementation experiments showed that adding 1 g/L methionine increased production from 17.16 ± 1.67 mg/L to 26.56 ± 0.80 mg/L, confirming that enhancing SAM availability can effectively boost methylation efficiency. Taken together, BL21-OMT successfully demonstrated the feasibility of sakuranetin biosynthesis, but production remained constrained by limited SAM supply and substrate inhibition. Based on these findings, in the next cycle we introduced regulatory elements to optimize energy and SAM metabolism for improved yields.

# Cycle 1-2 BL21-OMT-YdaO

## Design

In BL21-OMT, sakuranetin production was found to be limited by the insufficient availability of the methyl donor SAM. To overcome this bottleneck, we designed a second-generation strain, BL21-OMT-YdaO, by introducing the YdaO riboswitch. YdaO, an ATP-sensing riboswitch from *Bacillus subtilis*, dynamically regulates downstream gene transcription in response to intracellular energy levels. When co-expressed with OMT, YdaO is expected to enhance the Met–SAM cycle, thereby improving SAM synthesis and recycling efficiency. This provides a more abundant supply of methyl donors and energy for the OMT-catalyzed methylation of naringenin, ultimately aiming to increase sakuranetin production beyond the level achieved in BL21-OMT.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img8.webp)

Figure: Role of the YdaO riboswitch in dynamic ATP homeostasis control.

## Build

To enhance the host’s metabolic energy supply and SAM cycle efficiency, we introduced the YdaO element into the BL21-OMT system. The YdaO coding sequence(BBa\_25Z8A0VX, Generalbiol, China) was synthesized and codon-optimized for *E. coli*, with restriction sites incompatible with RFC#10 removed. The promoter J23100 and RBS B0034 were used as cis-regulatory elements. Using PCR and overlap extension, the B0034-YdaO fragment was placed downstream of F7-OMT, creating a co-expression circuit of OMT and YdaO. The recombinant construct was cloned into the pSB1A3 vector via XbaI and SpeI restriction sites, generating plasmid pSB1A3-OMT-YdaO. This plasmid was sequentially transformed into *E. coli* DH5α and BL21 (DE3), and positive clones were verified by antibiotic selection and sequencing. The resulting engineered strain, BL21-OMT-YdaO, was preserved in 25% glycerol at –80 ℃ and routinely cultured in LB broth at 37 ℃ and 150 rpm.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img9.webp)

Figure: Construction of BL21-OMT-YdaO. (A) The plasmid map of pSB1A3-OMT-YdaO. (B) Agarose gel electrophoresis of YadO (125 bp，without vhb and ptxD).We hypothesize that the fragment may have migrated out of the expected range due to excessively high voltage during electrophoresis. (C) The gene circuit of BL21-OMT-YdaO.

## Test

To evaluate the effect of YdaO on sakuranetin production, BL21-OMT and BL21-OMT-YdaO were cultured in TB medium supplemented with 150 mg/L naringenin at 25 ℃ for 12 h. After fermentation, cultures were processed by methanol extraction, centrifugation, and 0.22 μm filtration. Sakuranetin concentration was quantified using the established HPLC method, and yields were compared between the strains.

![Test   Diagram](https://static.igem.wiki/teams/6029/engineering/img10.webp)

Figure: Enhanced sakuranetin production in BL21-OMT-YdaO compared with BL21-OMT. Due to our oversight, the fermentation time in this cycle did not reach the 24 hours set in Cycle 1-1; therefore, we only compared sakuranetin production after 12 hours, and we plan to repeat the experiment for the full 24 hours in the future.

The first-generation strain BL21-OMT produced 18.96 ± 3.06 mg/L sakuranetin, whereas the co-expression strain BL21-OMT-YdaO achieved a significantly higher yield of 36.10 ± 2.19 mg/L, representing an overall improvement of nearly 90%. These results demonstrate that the introduction of YdaO effectively enhanced the Met–SAM cycle and energy supply, thereby significantly improving sakuranetin biosynthesis efficiency.

## Learn

In the BL21-OMT-YdaO experiments, sakuranetin production increased significantly from 18.96 ± 3.06 mg/L in the first-generation BL21-OMT to 36.10 ± 2.19 mg/L, representing an improvement of nearly 90%. This demonstrated that the introduction of YdaO effectively enhanced the Met–SAM metabolic cycle, partially alleviating the limitation of methyl donor availability. However, we also learned that despite this improvement, the yield was still below the level required for therapeutic application. This indicates that boosting energy metabolism alone cannot fully resolve the SAM supply bottleneck.

# Cycle 1-3 BL21-OMT-YdaO-CysE

## Design

In BL21-OMT-YdaO, the introduction of YdaO significantly improved energy metabolism and the Met–SAM cycle, but limited SAM availability remained the major bottleneck in sakuranetin biosynthesis. To address this, in the third-generation strain we introduced the CysE gene, which encodes L-serine O-acetyltransferase, a key enzyme in the cysteine biosynthetic pathway. By reinforcing the cysteine → methionine → SAM metabolic route, overexpression of CysE can increase intracellular methionine levels and thereby enhance SAM synthesis. We designed a co-expression system of OMT + YdaO + CysE to simultaneously strengthen methyl cycle efficiency and methyl donor supply, aiming to maximize the methylation of naringenin and further boost sakuranetin production.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img11.webp)

Figure : Reaction schematic of the third-generation system, with the main enhancement highlighted in the dark-shaded box on the left(Sun et al., 2022).

## Build

To further enhance methionine and SAM biosynthesis, we introduced the CysE gene into the BL21-OMT-YdaO system. The CysE (L-serine O-acetyltransferase) coding sequence (BBa\_25MTY54W, Generalbiol, China) was synthesized and codon-optimized for *E. coli*, with RFC#10-incompatible restriction sites removed. The promoter J23100 and RBS B0034 were placed upstream as cis-regulatory elements. Using PCR and overlap extension, the CysE module was inserted downstream of the OMT-YdaO construct, generating a three-gene co-expression circuit (OMT + YdaO + CysE). The recombinant sequence was cloned into the pSB1A3 vector via XbaI and SpeI restriction sites to yield plasmid pSB1A3-OMT-YdaO-CysE. The plasmid was first transformed into *E. coli* DH5α, with positive clones confirmed by ampicillin resistance and sequencing, and subsequently introduced into BL21 (DE3) to obtain the third-generation strain BL21-OMT-YdaO-CysE. The engineered strain was preserved at –80 ℃ in 25% glycerol and routinely cultured in LB broth at 37 ℃ and 150 rpm.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img12.webp)

Figure: Construction of BL21-OMT-YdaO-CysE. (A) The plasmid map of pSB1A3-OMT-YdaO-CysE (B) Agarose gel electrophoresis of cysE (819 bp).

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img13.webp)

Figure: The gene circuit of BL21-OMT-YdaO-CysE(From version 1 to version 3).

## Test

To evaluate the effect of CysE co-expression on sakuranetin production, BL21-OMT, BL21-OMT-YdaO, and BL21-OMT-YdaO-CysE were cultured in TB medium supplemented with 150 mg/L naringenin at 25 ℃ for 12 h. After fermentation, cultures were processed by methanol extraction, centrifugation, and 0.22 μm filtration. Sakuranetin concentration was quantified using the established HPLC method.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img14.webp)

Figure: Comparison of sakuranetin production among different engineered strains (BL21-OMT, BL21-OMT-YdaO, and BL21-OMT-YdaO-CysE). Due to our oversight, the fermentation time in this cycle did not reach the 24 hours set in Cycle 1-1; therefore, we only compared sakuranetin production after 12 hours, and we plan to repeat the experiment for the full 24 hours in the future.

The three generations showed progressive improvements in sakuranetin production: the first-generation BL21-OMT produced 18.92 ± 2.58 mg/L; the second-generation BL21-OMT-YdaO increased the yield to 31.07 ± 2.77 mg/L (p < 0.01); and the third-generation BL21-OMT-YdaO-CysE further boosted production to 45.21 ± 3.75 mg/L (p < 0.001), representing improvements of approximately 139% and 45% over the first and second generations, respectively. These results clearly demonstrate that the introduction of CysE significantly enhanced methionine and SAM availability, effectively overcoming the methyl donor limitation and enabling higher-level sakuranetin biosynthesis.

## Learn

In the BL21-OMT-YdaO-CysE experiments, the third-generation strain achieved a significantly higher sakuranetin yield of 45.21 ± 3.75 mg/L, representing an improvement of ~139% compared with BL21-OMT (18.92 ± 2.58 mg/L) and ~45% compared with BL21-OMT-YdaO (31.07 ± 2.77 mg/L). These results demonstrate that the combined introduction of YdaO and CysE successfully reinforced methionine and SAM availability, effectively overcoming the methyl donor limitation. From this, we learned that in synthetic pathways, not only catalytic efficiency but also the balance of metabolic precursors and energy supply is critical for maximizing production. Nevertheless, sakuranetin synthesis in this system still relies on exogenous naringenin supplementation, which limits independence and sustainability in real applications. Therefore, in the next cycle, we aimed to construct a strain capable of endogenous naringenin biosynthesis (BL21-Nar) to further enhance the completeness and applicability of the system.

# Cycle 1-4 BL21-Nar

## Design

In the previous cycles, the introduction of F7-OMT, YdaO, and CysE significantly improved sakuranetin production. However, the system still relied on the supplementation of exogenous (2S)-naringenin, which not only increased cost but also led to substrate inhibition, limiting the independence and sustainability of the pathway.

To overcome this limitation, in Cycle 1-4 we designed the engineered strain BL21-Nar, capable of endogenous naringenin biosynthesis. The strategy was to introduce the key enzymes of the plant flavonoid biosynthetic pathway into *E. coli*:

* TAL (tyrosine ammonia-lyase): converts L-tyrosine into p-coumaric acid;
* 4CL (4-coumaroyl-CoA ligase): activates p-coumaric acid to p-coumaroyl-CoA;
* CHS (chalcone synthase) and CHI (chalcone isomerase): catalyze the formation of (2S)-naringenin.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img15.webp)

Figure: Endogenous biosynthetic pathway for naringenin production.

This design enables the host to synthesize naringenin directly from its endogenous amino acid pool, providing a continuous substrate supply for downstream methylation. By reducing dependence on exogenous substrate and avoiding inhibition effects at high concentrations, BL21-Nar establishes a solid foundation for subsequent integration with the OMT module to achieve “endogenous supply + simultaneous conversion” in a unified production system.

## Build

To achieve endogenous naringenin biosynthesis, we reconstructed the upstream plant flavonoid pathway in *E. coli* BL21. Specifically, the TAL, 4CL, CHS, and CHI genes (BBa\_25886WFT, Generalbiol, China) were synthesized, codon-optimized for *E. coli*, and engineered to remove restriction sites incompatible with RFC#10 standards. To ensure stable expression and minimize promoter interference, TAL/4CL and CHS/CHI were designed as two parallel operons, each driven by the constitutive J23100 promoter with B0034 RBS, and terminated with a B0015 terminator. The dual-operon construct was assembled into the pSB1A3 vector, allowing compatibility with downstream integration of the OMT module. The recombinant plasmid was first transformed into *E. coli* DH5α, with positive clones confirmed by ampicillin resistance and sequencing. It was then introduced into BL21 (DE3), yielding the engineered strain BL21-Nar, capable of producing (2S)-naringenin. The strain was preserved in 25% glycerol at –80 ℃, and routinely cultured in LB broth (supplemented with 100 μg/mL Amp) at 37 ℃ and 150 rpm.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img16.webp)

Figure: Construction of BL21-Nar. (A) The plasmid map of pSB1A3-Nar. (B) Agarose gel electrophoresis of TAL (654 bp). (C) Agarose gel electrophoresis of 4CL (1710 bp). (D) Agarose gel electrophoresis of CHS (1167 bp). (E) Agarose gel electrophoresis of CHI (666 bp). （F）The gene circuit of BL21-Nar

## Test

A 100 μL aliquot of frozen BL21-Nar stock was inoculated into 5 mL Amp⁺ LB and incubated overnight at 37 ℃, 150 rpm. The culture was transferred into 250 mL Erlenmeyer flasks containing 50 mL Amp⁺ LB, adjusted to an initial OD₆₀₀ = 0.1, and grown at 37 ℃, 180 rpm until OD₆₀₀ ≈ 1. Cells were harvested (8000 × g, 5 min) and resuspended in M9 medium (A510881, Sangon) supplemented with 0.4% glucose (G6500, Innochem), 1 mM MgSO₄ (A40004, Innochem), 50 μM CaCl₂ (A01289, Innochem), 340 mg/L thiamine (BP892, Fisher), and 5 g/L CaCO₃ (A05990, Innochem). Cultures were incubated at 25 ℃, 180 rpm for 48 h.

* Naringenin quantification: 1 mL broth + 1 mL ethyl acetate, vortex 2 min, centrifuge 10,000 × g, 5 min; collect the organic phase, dry overnight at 37 ℃, redissolve in 1 mL acetonitrile, and read at 290 nm. Concentrations were calculated from a 0–800 mg/L standard curve (A06896, Innochem).
* Glucose quantification: 1 mL broth centrifuged 10,000 × g, 5 min; supernatants were analyzed using a glucose assay kit (60408ES60, Yeasen). Samples were ultrasonicated on ice (power 300 W, 3–5 s on / 30 s off, 3–5 cycles). In a 96-well plate, 2.5 μL distilled water/standard/sample + 250 μL working solution per well; incubate at 37 ℃ for 10 min and read at 505 nm to determine glucose from the standard curve.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img17.webp)

Figure: Principle of glucose assay based on the GOD–POD coupled colorimetric reaction

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img18.webp)

Figure: Time-course of naringenin production and glucose depletion during fermentation

Under 25 ℃, 180 rpm conditions, time-course monitoring over 48 h showed that naringenin in BL21-Nar increased from 0.0045 ± 0.0018 g/L (0 h) to 0.4214 ± 0.0474 g/L (48 h), while glucose decreased from 39.88 ± 1.70 g/L to 15.48 ± 2.68 g/L, indicating a clear negative correlation between substrate consumption and product accumulation. These data confirm that a recombinant production strain capable of endogenous naringenin biosynthesis was successfully constructed. At present, this strain operates as an independent module; future work will integrate it with the sakuranetin pathway to achieve coupled “production + in situ conversion.”

## Learn

In this cycle, we successfully demonstrated that the engineered strain BL21-Nar is capable of stable endogenous naringenin biosynthesis. After 48 h of fermentation, naringenin accumulation reached 0.4214 ± 0.0474 g/L, while glucose concentration decreased from 39.88 g/L to 15.48 g/L, showing a clear negative correlation between substrate consumption and product accumulation. These results confirm the effective operation of the biosynthetic pathway but also reveal potential bottlenecks: the overall yield remains below expectation, and carbon utilization efficiency appears limited, suggesting that precursor supply or cellular energy balance may still require optimization. From this cycle, we learned that E. coli can indeed serve as a feasible chassis for naringenin production, providing a robust substrate supply for downstream modules. Moving forward, we will integrate BL21-Nar with the OMT-based pathway to achieve coupled naringenin production and methylation, thereby improving the overall yield of sakuranetin.

# Cycle 2 BL21-PLA2

## Design

In Alzheimer’s disease (AD) patients, the abundance of Bacteroides ovatus in the gut and the serum level of lysophosphatidylcholine (LPC) are both significantly reduced, suggesting that the B. ovatus–PLA2–LPC metabolic axis may play a protective role.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img19.webp)

Figure 20: Microbiota - derived lysophosphatidylcholine alleviates Alzheimer's disease pathology via suppressing ferroptosis(Zha et al., 2025).

To reconstruct this pathway within a synthetic biology framework, we designed BL21-PLA2: an engineered *E. coli* BL21 strain expressing PLA2 from B. ovatus, catalyzing the selective hydrolysis of PC → LPC, thereby elevating LPC levels and providing a second functional route for AD intervention.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img20.webp)

Figure：the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC)

## Build

The phospholipase A2 (PLA2) coding gene (BBa\_25ST8FLD, Generalbiol, China) was synthesized and codon-optimized for *E. coli*, with restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI removed to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector. The gene was cloned into pET28a(m) via the NdeI and XhoI restriction sites, generating the recombinant plasmid. The recombinant plasmid was transformed into *E. coli* DH5α. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin (Kana) and verified by sequencing (Qingke, Beijing), yielding the recombinant strain DH5α-PLA2. Recombinant plasmids were extracted using a plasmid extraction kit (DP103, Tiangen) and subsequently transformed into *E. coli* BL21 (DE3) to obtain the engineered strain BL21-PLA2. The strain was preserved at –80 ℃ in 25% (v/v) glycerol as a cryoprotectant. Culture conditions for the engineered strain were 37 ℃ and 150 rpm, with inoculation and expansion carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img21.webp)

Figure: Construction of BL21-PLA2. (A) The plasmid map of pET-28a(m)-PLA2. (B) Agarose gel electrophoresis of PLA2 (843 bp). (C) The gene circuit of BL21-PLA2.

## Test

### Verification of PLA2 Expression (Western blot)

The BL21-PLA2 strain was inoculated into 50 mL LB medium supplemented with kanamycin and cultured overnight. Cells were harvested, resuspended in PBS, and disrupted by ultrasonication (150 W, 1 s on/3 s off cycles, 20 min total). The lysate was mixed with 5× reducing sample buffer (4:1), denatured in boiling water for 15 min, and loaded onto a 15% SDS-PAGE gel at 120 V. Proteins were transferred onto a PVDF membrane, blocked with 5% non-fat milk for 1 h, and incubated overnight at 4 ℃ with rabbit anti-PLA2 primary antibody (1:1000). After washing, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:1000) for 1 h at room temperature. Bands were visualized using ECL reagent and a chemiluminescence imaging system.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img22.webp)

Figure: Western blot analysis of PLA2 expression in engineered strain

A distinct band at 30–42 kDa was observed in BL21-PLA2, while absent in the control strain, confirming that PLA2 was successfully expressed in the engineered strain

### LPC Production Assay

BL21 and BL21-PLA2 were inoculated (1:100) into LB medium with 100 μg/mL kanamycin and cultured overnight at 37 ℃, 150 rpm. About 1 mL of the culture was transferred into 50 mL M9 medium (0.2% glucose, 1 mM MgSO₄, 50 μM CaCl₂, 100 μg/mL kanamycin) and adjusted to an initial OD₆₀₀ of 0.1. At OD₆₀₀ ≈ 0.6, 0.5 mM IPTG and 1 mg/mL egg yolk lecithin were added, followed by incubation for 24 h. After fermentation, 1 mL culture broth was centrifuged (10,000 × g, 10 min), and the supernatant was filtered (0.22 μm PES). The filtrate was diluted 100-fold and analyzed with a competitive LPC ELISA kit. A standard curve was generated (A450 = –0.915 × log₁₀C + 4.051, R² = 0.98), and absorbance at 450 nm was used to calculate LPC concentration.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img23.webp)

The LPC standard curve showed strong linearity (R² = 0.98), ensuring accurate quantification. The control strain BL21 produced only 2.43 ± 1.28 μg/mL LPC, while BL21-PLA2 produced significantly higher levels, 74.23 ± 15.91 μg/mL (p < 0.01) .

## Learn

Western blot analysis confirmed successful PLA2 overexpression, and metabolic assays demonstrated that BL21-PLA2 produced markedly higher LPC levels compared with the control strain. These findings validate the feasibility of reconstructing the B. ovatus–PLA2–LPC pathway in *E. coli*, providing a solid engineering basis for enhancing LPC levels in the gut. Together with the sakuranetin pathway developed in Cycle 1, this dual-module design represents a promising multi-target strategy for Alzheimer’s disease intervention.

# Cycle 3-1 DH5α–pBAD–mRFP

## Design

In the safety suicide system, our first step was to test whether the pBAD promoter can serve as a reliable artificial switch. We placed the mRFP reporter gene downstream of pBAD, generating DH5α–pBAD–mRFP. If fluorescence remains low without arabinose but increases with higher arabinose concentrations, it demonstrates that pBAD has low background and strong inducibility, making it suitable for driving the suicide module.

## Build

We synthesized the pBAD promoter (K808000), RBS B0034, and the mRFP gene, with mRFP codon-optimized for *E. coli* and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The construct was cloned into the pSB1A3 vector between the XbaI and SpeI restriction sites, generating the recombinant plasmid pBAD–mRFP. The plasmid was then transformed into *E. coli* DH5α, and positive clones were selected on LB agar plates supplemented with 50 μg/mL ampicillin (Amp). Sequencing verification (Qingke, Beijing) confirmed the correct construct, resulting in the engineered strain DH5α–pBAD–mRFP. The strain was cultured in LB broth at 37 ℃ for inoculation and expansion.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img24.webp)

Figure: Construction of DH5α-pBAD-mRFP. (A) The plasmid map of pSB1A3-pBAD-mRFP. (B) Agarose gel electrophoresis of araC-pBAD (1210 bp). Since the pBAD fragment is relatively short, it was linked with the araC regulatory sequence. (C) The gene circuit of DH5α-pBAD-mRFP.

## Test

The engineered strain DH5α–pBAD–mRFP was inoculated at a 1:100 ratio into 5 mL LB medium with 50 μg/mL ampicillin, with varying concentrations of L-arabinose (0%, 0.05%, 0.2%, 0.5%) added. Cultures were incubated at 37 ℃ and 180 rpm. At designated time points (0 h, 2 h, 4 h, 8 h), 200 μL samples were taken, and fluorescence (Ex 584 nm/Em 607 nm) and OD600 were measured using a microplate reader. Normalized fluorescence was calculated as Fluorescence/OD600.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img25.webp)

Figure: Dose- and time-dependent induction of pBAD promoter activity by L-arabinose

Under 0% arabinose, the fluorescence/OD600 ratio remained consistently low (0 h: 11.19 ± 0.56 AU; 2 h: 7.30 ± 0.52 AU; 4 h: 11.06 ± 0.63 AU; 8 h: 9.90 ± 1.98 AU), indicating minimal leakage of the pBAD promoter. In contrast, with 0.05% arabinose, fluorescence increased significantly by 2 h (69.80 ± 5.25 AU) and continued rising at 4 h (274.62 ± 77.33 AU) and 8 h (451.47 ± 139.93 AU), showing clear time dependence. At 0.2% arabinose, induction was stronger, reaching 524.11 ± 56.77 AU at 4 h and 829.13 ± 62.16 AU at 8 h. At 0.5% arabinose, the induction was the highest, already reaching 630.96 ± 44.09 AU at 4 h and exceeding 1089.19 ± 108.40 AU at 8 h. Overall, the fluorescence ratio increased continuously over time and scaled with arabinose concentration, confirming strong time-dependent and dose-dependent induction.

## Learn

From this cycle, we confirmed that the pBAD promoter has low basal activity and strong dose-dependent inducibility. Without arabinose, expression remained near baseline, indicating minimal leakage. Upon induction, the fluorescence/OD600 ratio increased continuously with both time and arabinose concentration, demonstrating rapid and controllable induction. This validates pBAD as a reliable artificial switch, capable of precisely triggering downstream gene expression when required. Based on these findings, we are confident to place the T4 lysis module under pBAD control to achieve artificially regulated suicide of engineered bacteria.

# Cycle 3-2 DH5α–pBAD–T4 lysis

## Design

After confirming that the pBAD promoter has low leakage and strong inducibility, we placed T4 holin and T4 lysozyme (forming the T4 lysis module) downstream of pBAD, designing the pBAD–T4 lysis system. In this system, the addition of L-arabinose activates the promoter, induces T4 lysis expression, and triggers cell lysis, thereby enabling artificially controlled clearance of engineered bacteria. This serves as the first layer of the safety suicide system, ensuring that strains can be rapidly eliminated when needed.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img26.webp)

Figure: Design principle of the arabinose-inducible suicide system (replacement of mRFP with T4-lysis downstream of pBAD)

## Build

We placed T4 holin (BBa\_254YB8C8) and T4 lysozyme (BBa\_25L8X41M) downstream of pBAD–B0034, generating the pBAD–T4 lysis construct. The gene sequences were codon-optimized and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards. The construct was cloned into the pSB1A3 vector at the XbaI/SpeI sites, yielding the recombinant plasmid pBAD–T4 lysis. The plasmid was then transformed into *E. coli* DH5α, with positive clones selected on LB agar plates containing 50 μg/mL ampicillin. Sequencing verification confirmed the correct construct, resulting in the engineered strain DH5α–pBAD–T4 lysis.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img27.webp)

Figure: Construction of DH5α-pBAD-T4 lysis. (A) The plasmid map of pSB1A3-pBAD-T4 lysis. (B) Agarose gel electrophoresis of T4 Holin (657 bp). (C) Agarose gel electrophoresis of T4 Lysozyme (492 bp). (D) The gene circuit of DH5α-pBAD-T4 lysis.

## Test

The engineered strain DH5α–pBAD–T4 lysis and the control strain DH5α were cultured overnight in LB + Amp at 37 ℃ and 180 rpm. A 100 μL inoculum was transferred into 5 mL LB + Amp for further cultivation. At 4 h, 0.5 mM L-arabinose was added to the experimental group. OD600 was measured at 0 h, 2 h, 4 h, 8 h, and 20 h.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img28.webp)

Figure：Growth dynamics of DH5α and pBAD–T4 lysis strains after L-arabinose induction at 4 h

Before induction, both strains showed similar growth: OD600 was 0.36 ± 0.11 (control-DH5α) and 0.38 ± 0.08 (experimental-DH5α/pBAD-T4 lysis) at 0 h, and increased to 0.67 ± 0.08 and 0.65 ± 0.11 at 2 h, respectively. At 4 h, after arabinose addition, OD600 values were 1.11 ± 0.17 (control) and 0.86 ± 0.12 (experimental), with no significant difference. Subsequently, clear divergence was observed: at 8 h, the control strain continued to grow to 1.60 ± 0.15, whereas the experimental strain dropped sharply to 0.29 ± 0.16. By 20 h, the control reached 2.31 ± 0.22, while the experimental strain remained at only 0.19 ± 0.16, indicating almost complete growth loss. These results demonstrate that the pBAD–T4 lysis system rapidly triggered cell lysis after arabinose induction, while the control strain maintained normal growth.

## Learn

This cycle demonstrated that the pBAD–T4 lysis system rapidly induced cell lysis after arabinose addition, almost completely suppressing the growth of engineered strains, while the control strain proliferated normally. This confirms that the system is a reliable artificial safety switch for rapid clearance of engineered bacteria when required.

# Cycle 3-3 DH5α–pCspA–mRFP

## Design

To prevent accidental spread outside the host, we adopt the cold-inducible promoter pCspA as an environmental trigger: ON at low temperature, OFF at 37 °C. We will evaluate its temperature responsiveness and basal leakage across 16 °C, 25 °C, and 37 °C. A strong induction at low temperatures with near-baseline activity at 37 °C would validate pCspA as an environmental switch suitable for driving the subsequent suicide module.

## Build

We synthesized the cold-inducible promoter pCspA (K4987003), RBS B0034, and the mRFP reporter. The mRFP was codon-optimized for *E. coli* and edited to remove EcoRI, XbaI, SpeI, and PstI sites to comply with RFC#10 (Genewiz, USA). The pCspA–B0034–mRFP cassette was cloned into the pSB1A3 vector via XbaI/SpeI, yielding pSB1A3–pCspA–mRFP. The recombinant plasmid was transformed into *E. coli* DH5α; positive colonies were selected on LB agar containing 50 μg/mL ampicillin and verified by sequencing (Qingke, Beijing), resulting in the engineered strain DH5α–pCspA–mRFP. The strain was routinely cultured at 37 °C in LB + Amp for subsequent temperature-response assays.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img29.webp)

Figure: Construction of DH5α-pCspA-mRFP. (A) The plasmid map of pSB1A3-pCspA-mRFP. (B) Agarose gel electrophoresis of pCspA-mRFP (738 bp); since the pCspA promoter is too short, the mRFP reporter was placed downstream for validation. (C)The gene circuit of DH5α-pCspA-mRFP.

## Test

The engineered strain DH5α–pCspA–mRFP was activated overnight and inoculated at 1:100 into LB + Amp medium. Cultures were incubated at 16 ℃, 25 ℃, and 37 ℃ with shaking (180 rpm, 12 h). At 2 h, 6 h, and 12 h, 200 μL samples were collected, and fluorescence (Ex 584 nm/Em 607 nm) and OD600 were measured using a microplate reader. Normalized fluorescence (fluorescence/OD600) was calculated.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img30.webp)

Figure: Temperature-dependent activation of pCspA promoter driving mRFP expression

At 16 ℃, fluorescence/OD600 rose rapidly over time, from 32.77 ± 14.58 AU at 2 h to 168.20 ± 20.80 AU at 6 h, reaching 270.84 ± 19.36 AU at 12 h, showing strong cold induction. At 25 ℃, induction was moderate, reaching 240.04 ± 14.51 AU at 12 h, slightly lower than at 16 ℃. At 37 ℃, fluorescence remained near background, with only 67.92 ± 22.24 AU at 12 h.These results demonstrate that the pCspA promoter strongly drives gene expression at low temperatures but remains nearly inactive at 37 ℃, showing a clear “ON at low temperature, OFF at high temperature” profile.

## Learn

This cycle clearly demonstrated that the pCspA promoter strongly induces gene expression at 16 ℃ while remaining nearly inactive at 37 ℃. This confirms its suitability as an environmental trigger: when engineered bacteria escape the host and encounter low-temperature conditions (16 ℃), pCspA can activate the suicide module, while inside the host at 37 ℃ it stays silent to preserve normal function. Therefore, we conclude that pCspA is a reliable environmental switch and will be combined with the T4 lysis module in the next step to establish a cold-inducible suicide system.

# Cycle 3-4 DH5α–pCspA–T4 lysis

## Design

In this cycle, we used the cold-inducible promoter pCspA (activated at 16 ℃, nearly inactive at 37 ℃) to drive the expression of T4 holin and T4 lysozyme, constructing DH5α–pCspA–T4 lysis. This design allows engineered bacteria to undergo self-lysis automatically under low-temperature conditions (16 ℃) outside the host, while remaining silent at body temperature (37 ℃) to preserve normal function.

![Design Diagram](https://static.igem.wiki/teams/6029/engineering/img31.webp)

Figure: Design principle of the cold-inducible suicide system (replacement of mRFP with T4-lysis downstream of pCspA)

## Build

We placed the T4 holin (BBa\_254YB8C8) and T4 lysozyme (BBa\_25L8X41M) genes downstream of pCspA–B0034 to form the cold-inducible suicide module. The sequences were codon-optimized and modified to remove EcoRI, XbaI, SpeI, and PstI sites to meet RFC#10 standards. The construct was cloned into the pSB1A3 vector via XbaI/SpeI restriction sites, generating the plasmid pSB1A3–pCspA–T4 lysis. The recombinant plasmid was transformed into *E. coli* DH5α, positive clones were selected on LB agar plates containing 50 μg/mL ampicillin, and sequencing confirmed the correct construct, yielding the engineered strain DH5α–pCspA–T4 lysis.

![Build Diagram](https://static.igem.wiki/teams/6029/engineering/img32.webp)

Figure: The plasmid map of pSB1A3-pCspA-T4 lysis.

# Test

The engineered strain DH5α–pCspA–T4 lysis and the control strain DH5α were cultured overnight, and 100 μL of each was inoculated into 5 mL LB medium with 50 μg/mL ampicillin. Cultures were incubated at 16 ℃ with shaking at 180 rpm. At 0 h, 2 h, 4 h, and 8 h, 200 μL samples were collected, and OD600 was measured to compare the growth of the two strains.

![Test Diagram](https://static.igem.wiki/teams/6029/engineering/img33.webp)

Figure：Growth inhibition of DH5α/pCspA–T4 lysis at 16 ℃ compared with wild-type DH5α

At 0 h, OD600 values were 0.21 ± 0.03 for the control and 0.19 ± 0.05 for the engineered strain, showing no difference. As incubation proceeded, the control strain grew normally, reaching 0.36 ± 0.04 (2 h), 0.60 ± 0.07 (4 h), and 0.83 ± 0.06 (8 h). In contrast, the engineered strain exhibited strong growth inhibition, with OD600 remaining low at 0.22 ± 0.06 (2 h), 0.31 ± 0.06 (4 h), and 0.34 ± 0.08 (8 h). These results demonstrate that the pCspA–T4 lysis system significantly restricted bacterial growth at 16 ℃, while the control strain proliferated normally, confirming the effectiveness of this cold-inducible suicide module.

## Learn

This cycle demonstrated that the pCspA–T4 lysis system effectively suppresses growth at 16 ℃ . This confirms its role as a reliable environment-triggered safety switch, complementing the pBAD–T4 lysis system to build a dual-layer safeguard and further reduce the risk of engineered strain escape.

# Reference

[1] Sun, Q., Gao, S., Yu, S., Zheng, P., & Zhou, J. (2022). Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance. Synthetic and systems biotechnology, 7(4), 1117-1125.

[2] Zha, X., Liu, X., Wei, M., Huang, H., Cao, J., Liu, S., ... & Zhang, C. (2025). Microbiota-derived lysophosphatidylcholine alleviates Alzheimer’s disease pathology via suppressing ferroptosis. Cell metabolism, 37(1), 169-186.

[3] Couse, N. L. (1968). Control of lysis of T4-infected Escherichia coli. Journal of Virology, 2(3), 198-207.

[4] <https://registry.igem.org/parts/bba-k808000>

[5] [https://parts.igem.org/Part:BBa\_K4987003](https://parts.igem.org/Part%3ABBa_K4987003)

{% 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 %}

![](https://static.igem.wiki/teams/6029/common/content-title.webp)

* [Construction of (2S)-Sakuranetin-Producing Strains](#section-1)
  + [Construction of F7-OMT and YdaO Co-Expression Strain](#section-2)
    - [Construction of Methionine-Supplemented Strain](#section-3)
      * [Analysis of (2S)-Sakuranetin Production](#section-4)
        + [Effect of Methionine Supplementation on Sakuranetin Biosynthesis](#section-5)
          - [Construction of Naringenin-Producing Strains](#section-6)
            * [Naringenin Production Assay](#section-7)
              + [Construction of PLA2-Overexpressing Strain](#section-8)
                - [Western blot verification of PLA2 expression](#section-9)
                  * [PLA2 Activity Assay](#section-10)
                    + [Construction and Characterization of Arabinose-Inducible Reporter Strain](#section-11)
                      - [Construction and Testing of Arabinose-Inducible Suicide System](#section-12)
                        * [Construction and Characterization of Cold-Inducible Reporter Strain](#section-13)
                          + [Construction and Testing of Cold-Inducible Suicide System](#section-14)
                            - [Statistical Analysis](#section-15)
                              * [Related kit instructions:](#section-16)

# Construction of (2S)-Sakuranetin-Producing Strains

The F7-OMT gene (BBa\_259M31WV, Generalbiol, China) was synthesized and codon-optimized for *E. coli*. Restriction sites EcoRI, XbaI, SpeI, and PstI were removed to comply with RFC#10 standards. The promoter–RBS cassette J23100-B0034 was used as a cis-regulatory element and cloned into the pSB1A3 vector via XbaI and SpeI sites. *E. coli* BL21 (DE3) (D1009S) and DH5α (D0351) competent cells were purchased from Beyotime (China). Recombinant plasmids were transformed into *E. coli* DH5α, plated on LB agar plates (1.5% agar) supplemented with 100 μg/mL ampicillin, and positive clones were verified by sequencing (Qingke, Beijing). The resulting recombinant strain was designated DH5α-OMT. Plasmids were extracted using a commercial plasmid extraction kit (DP103, Tiangen) and subsequently transformed into *E. coli* BL21 (DE3), generating strain BL21-OMT. Strains were stored at −80 ℃ in 25% (v/v) glycerol. For cultivation, strains were grown at 37 ℃ and 150 rpm in LB broth (G3102, Servicebio, China) containing 100 μg/mL ampicillin.

# Construction of F7-OMT and YdaO Co-Expression Strain

The YdaO gene (BBa\_25Z8A0VX, Generalbiol, China) was synthesized, codon-optimized for *E. coli*, and restriction sites avoided by RFC#10 were removed. The J23100-B0034 cassette was placed upstream, and the construct was cloned into pSB1A3 via XbaI and SpeI. PCR and overlap extension were applied to place B0034-YdaO downstream of F7-OMT, yielding the F7-OMT–YdaO co-expression sequence, which was also cloned into pSB1A3. Recombinant plasmids were transformed into *E. coli* DH5α and BL21, plated on LB agar containing 100 μg/mL ampicillin, and verified by sequencing. The resulting strain was designated BL21-OMT-YdaO. Strains were stored at −80 ℃ in 25% (v/v) glycerol and cultured under the same conditions as above.

# Construction of Methionine-Supplemented Strain

To enhance methionine production in *E. coli*, the *cysE* gene encoding L-serine O-acetyltransferase (BBa\_25MTY54W, Generalbiol, China) was synthesized and codon-optimized. The J23100-B0034 cassette was placed upstream of *cysE*, and PCR with overlap extension was used to insert J23100-B0034-*cysE* downstream of the OMT-YdaO module. The OMT–YdaO–CysE operon was cloned into pSB1A3 via XbaI and SpeI. Following transformation and selection, the recombinant strain was designated BL21-OMT-YdaO-CysE. Strains were stored and cultured under the same conditions described above.

# Analysis of (2S)-Sakuranetin Production

Overnight cultures of engineered strains were prepared in LB medium with 100 μg/mL ampicillin at 37 ℃, 150 rpm. BL21-OMT and BL21-OMT-YdaO were inoculated into Terrific Broth (TB; BL1176A, Biosharp) supplemented with 100 μg/mL ampicillin. Cultures were incubated at 37 ℃, 180 rpm until OD600 = 0.6, followed by supplementation with 150 mg/L (2S)-naringenin (A06896, Innochem). Cultures were then shifted to 25 ℃ and incubated for 24 h. Samples (1 mL) were collected, mixed with an equal volume of methanol, centrifuged at 12,000 × g for 5 min, and filtered through a 0.22 μm nylon membrane (110414017, BKMAN). HPLC analysis was conducted using an Agilent 1200 system with a C18 column (4.6 × 250 mm, 5 μm) at 290 nm. The mobile phases consisted of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). The gradient program was as follows: 0–10 min, 10–60% B; 10–20 min, 40–80% B; 20–25 min, 80–10% B.

# Effect of Methionine Supplementation on Sakuranetin Biosynthesis

To assess the effect of methyl donors on sakuranetin biosynthesis, BL21-OMT was cultured in TB with ampicillin under the same conditions as above until OD600 = 0.6. After 3 h at 37 ℃, 180 rpm, cultures were supplemented with 150 mg/L (2S)-naringenin and 1 g/L methionine (A49392, Innochem), shifted to 25 ℃, and incubated for 12 h. Samples were processed as described above and analyzed for sakuranetin content.

# Construction of Naringenin-Producing Strains

The *TAL* BBa\_25USFVFA, *4CL* BBa\_256H06VX, *CHS* BBa\_25NCLJJ7, and *CHI* BBa\_25I7VX04 genes (Generalbiol, China) were synthesized, codon-optimized, and modified to remove RFC#10-restricted sites. J23100-B0034 promoters were used to independently drive *TAL/4CL* and *CHS/CHI* expression, with T0015 terminators inserted to prevent promoter interference. Constructs were cloned into pSB1A3 via XbaI and SpeI. Recombinant plasmids were transformed into *E. coli* BL21, generating strain BL21-Nar. Strains were stored and cultured as described above.

# Naringenin Production Assay

Frozen cultures (100 μL) were inoculated into 5 mL LB with ampicillin and incubated overnight at 37 ℃, 150 rpm. Pre-cultures were inoculated into 50 mL LB with ampicillin in 250 mL flasks (initial OD600 = 0.1) and grown to OD600 = 1 at 37 ℃, 180 rpm. Cells were harvested by centrifugation (8,000 × g, 5 min) and resuspended in M9 medium (A510881, Sangon Biotech) supplemented with 0.4% glucose, 3 mM L-tyrosine, 1 mM MgSO4, 50 μM CaCl2, and 340 mg/L thiamine. Cultures were incubated at 25 ℃, 180 rpm for 48 h. Supernatants were extracted with an equal volume of ethyl acetate, centrifuged, and the organic phase collected, dried at 37 ℃ overnight, and resuspended in 1 mL acetonitrile. Absorbance at 290 nm was measured using a plate reader (FlexStation 3, Molecular Devices, USA). Concentrations were calculated against a naringenin standard curve (0–800 mg/L). Glucose levels were determined using a glucose detection kit (60408ES60, Yeasen, China). For more information on the glucose detection kit, please refer to the following link: <https://www.yeasen.com/products/detail/1904741509662253058.>

# Construction of PLA2-Overexpressing Strain

The *PLA2* gene (BBa\_25ST8FLD, Generalbiol, China) was synthesized and codon-optimized for *E. coli.* Restriction sites incompatible with RFC#10 and pET28a(m) cloning were removed. The optimized sequence was cloned into the pET28a(m) vector using NdeI and XhoI restriction sites and transformed into *E. coli* DH5α. Positive clones were selected on LB agar plates containing 100 μg/mL kanamycin and verified by sequencing. Plasmids from verified clones were extracted and subsequently transformed into *E. coli* BL21 (DE3), generating the BL21-PLA2 strain. The engineered strain was stored at -80℃ with 25% (v/v) glycerol and cultured in LB broth supplemented with 100 μg/mL kanamycin.

# Western blot verification of PLA2 expression

The PLA2-overexpressing engineered strain was inoculated into 50 mL LB medium supplemented with kanamycin and cultured overnight. The next day, bacterial cultures were collected and centrifuged at 10,000 × g for 1 min. The cell pellet was resuspended in PBS and disrupted by ultrasonication (150 W, 1 s on/3 s off cycles, for a total of 20 min). The resulting lysate was transferred into a new centrifuge tube as the intracellular protein sample. Protein solutions were mixed with 5× reducing sample buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 min. Proteins were separated on 15% SDS-PAGE gels at 120 V and subsequently transferred onto a PVDF membrane under cold conditions. The membrane was blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by overnight incubation at 4 ℃ with a rabbit anti-PLA2 primary antibody (1:1000; commonly sourced from Abcam or Cayman). After three 10 min washes with TBST, the membrane was incubated at room temperature for 1 h with HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:1000 dilution, A0208, Beyotime). Following another three washes with TBST (10 min each), ECL reagents A and B were mixed at a 1:1 ratio. The washed PVDF membrane was placed on absorbent paper to remove excess liquid and then covered with the prepared ECL solution for 1 min to ensure full coverage. Excess liquid was gently removed, and the membrane was exposed using a chemiluminescence imaging system under preset parameters.

# PLA2 Activity Assay

Overnight cultures of BL21-PLA2 were inoculated at a 1:100 ratio into M9 medium supplemented with 0.2% (w/v) glucose, 1 mM MgSO4, 50 μM CaCl2, and 100 μg/mL kanamycin. Cultures were grown at 37℃ with shaking until OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG and 1 mg/mL lecithin (L305002, Aladdin), and cultures were incubated for an additional 24 h.

After incubation, 1 mL of culture was centrifuged at 10,000 × g for 10 min, and the supernatant was filtered through a 0.22 μm PES membrane. The filtrate was diluted 100-fold with PBS, and lysophosphatidylcholine (LPC) levels were quantified using a commercial ELISA kit. Absorbance at 450 nm was measured with a microplate reader (FlexStation 3, Molecular Devices, USA) to determine LPC concentration.

# Construction and Characterization of Arabinose-Inducible Reporter Strain

The PBAD promoter (K808000), RBS B0034, and mRFP sequence were synthesized (Genewiz, USA), codon-optimized, and cloned into the pSB1A3 vector. The recombinant plasmid was transformed into *E. coli* DH5α, and colonies were screened on LB agar plates containing 50 μg/mL ampicillin. Positive clones were verified by sequencing and cultured in LB medium with 50 μg/mL ampicillin. Fluorescence (Ex 584 nm, Em 607 nm) and OD600 were measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) after induction with varying concentrations of L-arabinose.

# Construction and Testing of Arabinose-Inducible Suicide System

For the suicide system, the PBAD-B0034 regulatory module was placed upstream of the T4 holin and T4 lysozyme (T4 lysis) genes. The coding sequences were codon-optimized for *E. coli* and cloned into pSB1A3. Recombinant strains were grown in LB medium containing 50 μg/mL ampicillin. Cultures were induced with 0.5 mM L-arabinose, and OD600 was measured over time to evaluate cell lysis efficiency.

# Construction and Characterization of Cold-Inducible Reporter Strain

The cold-shock promoter PcspA (K4987003) was synthesized and cloned upstream of mRFP in the pSB1A3 vector. The mRFP sequence was codon-optimized and verified by sequencing. Recombinant strains were cultured in LB medium supplemented with 50 μg/mL ampicillin. Inductions were performed at different temperatures, and fluorescence intensity was normalized to OD600. Fluorescence was detected using a plate reader (Thermo Fisher Scientific).

# Construction and Testing of Cold-Inducible Suicide System

For the cold-inducible suicide system, the PcspA promoter was cloned upstream of the T4 holin and T4 lysozyme genes, which were codon-optimized and inserted into the pSB1A3 plasmid. Recombinant strains were grown in LB medium containing 50 μg/mL ampicillin. Cultures were incubated at 16 ℃ under shaking conditions, and OD600 was measured at different time points to assess lysis activity.

# Statistical Analysis

Data were analyzed and visualized using GraphPad Prism. Results are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test for multiple comparisons and Student’s t-test for two-group comparisons. A p-value < 0.05 was considered statistically significant.

# Related kit instructions:

Lysophosphatidylcholine (LPC) Enzyme-Linked Immunosorbent Assay (ELISA) Kit

Lysophosphatidylcholine (LPC) Enzyme-Linked Immunosorbent Assay (ELISA) Kit.pdf

0.6MB

[Download](https://static.igem.wiki/teams/6029/experiments/lysophosphatidylcholine-lpc-enzyme-linked-immunosorbent-assay-elisa-kit.pdf)

Phosphatidylcholine (PC) Detection Kit

Phosphatidylcholine (PC) Detection Kit.pdf

0.3MB

[Download](https://static.igem.wiki/teams/6029/experiments/phosphatidylcholine-pc-detection-kit.pdf)

{% 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 %}

![](https://static.igem.wiki/teams/6029/common/content-title.webp)

* [Abstract](#section-1)
  + [System 1：Sakuranetin production system](#section-2)
    - [HPLC analysis of naringenin and sakuranetin](#section-3)
      * [Objective：](#subsection-4)
      * [Method：](#subsection-5)
      * [Result：](#subsection-6)
      * [Conclusion：](#subsection-7)
    - [BL21-OMT：F7-OMT promotes the production of sakuranetin](#section-8)
      * [Objective：](#subsection-9)
      * [Method：](#subsection-10)
      * [Result：](#subsection-11)
      * [Conclusion：](#subsection-12)
    - [Effect of naringenin concentration on sakuranetin production](#section-13)
      * [Objective：](#subsection-14)
      * [Method：](#subsection-15)
      * [Result：](#subsection-16)
      * [Conclusion ：](#subsection-17)
    - [Methionine supplementation promotes sakuranetin production](#section-18)
      * [Objective：](#subsection-19)
      * [Method：](#subsection-20)
      * [Result：](#subsection-21)
      * [Conclusion：](#subsection-22)
    - [BL21-*OMT*-*YdaO*：Improved Sakuranetin production via *YdaO* co-expression](#section-23)
      * [Objective：](#subsection-24)
      * [Method：](#subsection-25)
      * [Result：](#subsection-26)
      * [Conclusion：](#subsection-27)
    - [BL21-*OMT-YdaO-CysE*: Further enhancement of Sakuranetin production by *CysE* co-expression](#section-28)
      * [Objective：](#subsection-29)
      * [Method：](#subsection-30)
      * [Result：](#subsection-31)
      * [Conclusion：](#subsection-32)
    - [Analysis of naringenin production](#section-33)
      * [Objective：](#subsection-34)
      * [Method：](#subsection-35)
      * [Result：](#subsection-36)
      * [Conclusion：](#subsection-37)
    - [System 2： LPC Production System](#section-38)
      * [LPC Degradation Analysis](#section-39)
        + [Objective：](#subsection-40)
        + [Method：](#subsection-41)
        + [Result：](#subsection-42)
        + [Conclusion：](#subsection-43)
      * [System 3： Safety Suicide System](#section-44)
        + [DH5α-pBAD-mRFP：Validation of pBAD promoter activity for inducible suicide system](#section-45)
          - [Objective：](#subsection-46)
          - [Method：](#subsection-47)
          - [Result：](#subsection-48)
          - [Conclusion：](#subsection-49)
        + [DH5α-pBAD–T4 lysis: Arabinose-inducible suicide system](#section-50)
          - [Objective：](#subsection-51)
          - [Method：](#subsection-52)
          - [Result：](#subsection-53)
          - [Conclusion：](#subsection-54)
        + [DH5α-pCspA–mRFP: Validation of cold-inducible reporter activity](#section-55)
          - [Objective：](#subsection-56)
          - [Method：](#subsection-57)
          - [Result：](#subsection-58)
          - [Conclusion：](#subsection-59)
        + [DH5α-pCspA–T4 lysis: Validation of cold-inducible suicide system](#section-60)
          - [Objective：](#subsection-61)
          - [Method：](#subsection-62)
          - [Result：](#subsection-63)
          - [Conclusion：](#subsection-64)
        + [Reference](#section-65)

# Abstract

We engineered a multi-target probiotic chassis with three functional systems to enhance efficacy and ensure biosafety in Alzheimer’s disease intervention. System 1 (Sakuranetin production): Expression of F7-OMT boosted sakuranetin to 50.21 ± 2.67 mg/L, further enhanced to 45.21 ± 3.75 mg/L through *YdaO* and CysE co-expression, with BL21-Nar supplying 0.42 g/L naringenin as precursor. System 2 (LPC production): Overexpression of PLA2 yielded a clear protein band (~30–42 kDa) and increased LPC from 2.43 ± 1.28 μg/mL in controls to 74.23 ± 15.91 μg/mL. System 3 (Dual-layer kill switches): The arabinose-inducible pBAD–T4 lysis reduced OD600 to 0.19 at 20 h, while the cold-inducible pCspA–T4 lysis suppressed growth at 16 ℃ (OD600 0.34 vs. 0.83 in control). Together, these three systems establish a probiotic platform capable of high-level metabolite production with robust “user-triggered + environment-triggered” containment, balancing efficacy and biosafety.

![Abstract Diagram](https://static.igem.wiki/teams/6029/results/img1.webp)

Figure: three functional systems to enhance efficacy and ensure biosafety in Alzheimer’s disease intervention

# System 1：Sakuranetin production system

We established a sakuranetin production system using naringenin as the substrate and progressively optimized it through three iterative generations. In the first-generation strain BL21-OMT, the introduction of *F7-OMT* significantly increased sakuranetin production from 0.65 ± 0.05 mg/L in the control to 49.46 ± 9.53 mg/L. Substrate optimization revealed that 150 mg/L naringenin yielded the highest production (50.21 ± 2.67 mg/L), and supplementation with 1 g/L methionine further enhanced production by approximately 55%. In the second-generation strain BL21-*OMT*-*YdaO*, co-expression of *ydaO* strengthened energy supply and the Met–SAM cycle, increasing production from 18.92 ± 2.58 mg/L to 31.07 ± 2.77 mg/L . The third-generation strain BL21-OMT-*YdaO*-CysE further introduced CysE to reinforce the cysteine-to-methionine pathway and boost SAM availability, resulting in an additional increase to 45.21 ± 3.75 mg/L. In parallel, BL21-Nar accumulated 0.42 ± 0.05 g/L naringenin during 48 h fermentation, providing a stable substrate supply for sakuranetin synthesis. Overall, through stepwise optimization across the first, second, and third generations (*F7-OMT* → *ydaO* → *CysE*), the system achieved more stable and efficient sakuranetin production, laying the foundation for future integration of endogenous naringenin supply with simultaneous conversion into sakuranetin in a single streamlined system.

# HPLC analysis of naringenin and sakuranetin

## Objective：

To establish and validate a high-performance liquid chromatography (HPLC) method for the qualitative and quantitative analysis of the substrate (2S)-naringenin and the product (2S)-Sakuranetin, providing a reliable analytical approach for subsequent yield optimization(Sun et al., 2022).

## Method：

The *F7-OMT* gene (BBa\_259M31WV, Generalbiol, China) was synthesized and codon-optimized for expression in *E. coli.* EcoRI, XbaI, SpeI, and PstI restriction sites were removed to meet the RFC#10 standard. The promoter J23100 and RBS B0034 were used as cis-regulatory elements, and the gene fragment was cloned into the pSB1A3 vector via XbaI and SpeI restriction sites. Competent *E. coli* BL21 (DE3) (D1009S) and DH5α (D0351) cells were purchased from Beyotime (China). The recombinant plasmid was first transformed into *E. coli* DH5α. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing), generating the recombinant strain DH5α-OMT. The recombinant plasmid was then extracted using a plasmid extraction kit (DP103, Tiangen) and transformed into *E. coli* BL21 (DE3) to obtain the engineered strain BL21-OMT. Strains were stored at –80 ℃ in 25% (v/v) glycerol. The engineered strains were cultured at 37 ℃ and 150 rpm in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL ampicillin for inoculation and amplification.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img2.webp)

Figure: Construction of BL21-OMT. (A) The plasmid map of pSB1A3-OMT. (B) Agarose gel electrophoresis of *F7-OMT* (1056 bp). (C) The gene circuit of BL21-OMT.

A 1:100 inoculum of the engineered strain BL21-OMT was transferred into LB broth containing 100 μg/mL ampicillin and cultured overnight at 37 ℃, 150 rpm for activation. Activated cells were inoculated into TB medium (BL1176A, Biosharp) supplemented with Amp. The culture was incubated at 37 ℃, 180 rpm for 3–4 h until OD600 = 0.6, followed by the addition of 150 mg/L naringenin (A06896, Inonochem). The culture temperature was then shifted to 25 ℃ and fermentation continued for 24 h. After fermentation, 1 mL of the culture broth was mixed with 1 mL methanol, centrifuged at 12,000 × g for 5 min, and the supernatant filtered through a 0.22 μm nylon organic-phase filter (110414017, BKMAN). Samples were analyzed by HPLC (Agilent 2100) equipped with an Agilent C18 column (4.6 mm × 250 mm, 5 μm), with detection at 290 nm. The mobile phases were water containing 0.1% TFA (phase A) and acetonitrile containing 0.1% TFA (phase B), with a flow rate of 1 mL/min, using the following gradient: 0–10 min: 10% → 60% B; 10–20 min: 40% → 80% B; 20–25 min: 80% → 10% B. At this stage, phase B was initiated at 40% (instead of 60%) to optimize the resolution of the target component and avoid peak overlap.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img3.webp)

Figure：High performance liquid chromatography（HPLC）to detect naringenin and sakuranetin

## Result：

HPLC analysis revealed two distinct and stable peaks at 12.4 min and 24.5 min, corresponding to the substrate naringenin and the product sakuranetin, respectively. Comparison with standards confirmed the peak identities, validating the accuracy of substrate conversion and product formation. Both peaks were sharp and symmetrical with stable baselines, indicating excellent separation performance, with no significant tailing or co-elution interference, ensuring reliable and reproducible detection results.

Based on the standard curve fitting formula, peak areas can be converted into concentration values, enabling quantitative analysis of naringenin consumption and sakuranetin production under various conditions.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img4.webp)

Figure: HPLC chromatograms of naringenin and sakuranetin

## Conclusion：

This HPLC method enables sensitive and stable detection of both naringenin and sakuranetin, accurately reflecting the dynamic changes in substrate consumption and product generation. It provides a reliable analytical tool for comparing sakuranetin production across different strains, substrate concentrations, and fermentation conditions.

# BL21-OMT：F7-OMT promotes the production of sakuranetin

## Objective：

After a stable and reliable HPLC method was established, the effect of flavonoid 7-O-methyltransferase (F7-OMT) gene expression on sakuranetin production was evaluated. In this reaction, naringenin and S-adenosylmethionine (SAM) were converted into sakuranetin and S-adenosylhomocysteine (SAH) under the catalysis of *F7-OMT*.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img5.webp)

## Method：

The control strain BL21 and the engineered strain BL21-OMT carrying the *F7-OMT* gene were inoculated into TB medium supplemented with 150 mg/L naringenin and fermented at 25 ℃ for 24 h. After fermentation, culture samples were treated with methanol, centrifuged, and filtered through a 0.22 μm organic membrane filter. Product peak areas were then detected at 290 nm using the previously established HPLC method, and the sakuranetin concentration was calculated.

## Result：

Sakuranetin production was compared between the engineered strain BL21-OMT and the control strain BL21. The control strain produced only a very low level of sakuranetin (0.65 ± 0.05 mg/L), whereas the BL21-OMT strain exhibited a markedly higher yield, reaching 49.46 ± 9.53 mg/L—an approximately 76-fold increase on average.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img6.webp)

Figure：Sakuranetin production in BL21 and BL21-OMT strains after 24 h fermentation

## Conclusion：

The expression of *F7-OMT* greatly enhanced sakuranetin production in the engineered strain, confirming its key role in sakuranetin biosynthesis. However, the current yield (49.46 ± 9.53 mg/L) is still insufficient for our needs; therefore, subsequent work will focus on optimizing experimental conditions to further improve sakuranetin production.

# Effect of naringenin concentration on sakuranetin production

## Objective：

Sakuranetin can be produced from naringenin under the catalysis of flavonoid *F7-OMT*. Since naringenin is an essential precursor, we aimed to investigate the effect of its concentration on sakuranetin production in the engineered strain BL21-OMT.

## Method：

The engineered strain BL21-OMT was inoculated into TB medium supplemented with 100, 150, and 300 mg/L naringenin and fermented at 25 ℃ for 24 h. Sakuranetin concentration was measured using the same analytical procedure as described above.

## Result：

Sakuranetin production by the engineered strain varied significantly under different naringenin concentrations (ANOVA, F = 21.09, p = 0.0019 < 0.05). The highest yield was obtained at 150 mg/L naringenin (50.21 ± 2.67 mg/L). At 100 mg/L, the yield was slightly lower (39.19 ± 4.84 mg/L) but not significantly different from the 150 mg/L group. In contrast, at the high concentration of 300 mg/L naringenin, sakuranetin production decreased significantly to 26.47 ± 5.45 mg/L.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img7.webp)

Figure: Sakuranetin production of BL21-OMT under different naringenin concentrations

## Conclusion ：

Substrate concentration had a significant effect on sakuranetin production in the *F7-OMT* engineered strain, with 150 mg/L naringenin giving the highest yield. Although no significant difference was observed between the 100 mg/L and 150 mg/L groups, this may be due to large variation within the group or the small sample size, which reduced statistical power. At 300 mg/L, the yield dropped significantly, likely because of substrate inhibition or toxic stress affecting cell metabolism and enzymatic conversion. Therefore, 150 mg/L naringenin was used as the standard culture condition for subsequent experiments on sakuranetin production.

# Methionine supplementation promotes sakuranetin production

## Objective：

Building on the investigation of substrate concentration, we further evaluated the effect of exogenous methionine (Met) supplementation on sakuranetin production in the engineered strain BL21-OMT. Since Met is converted into S-adenosylmethionine (SAM) under the catalysis of MetK, and SAM serves as the methyl donor in the *F7-OMT*–mediated reaction, this experiment aimed to verify the promoting role of Met in sakuranetin biosynthesis.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img8.webp)

Figure: Met-methionine is converted into S-adenosylmethionine (SAM) under the catalysis of MetK

## Method：

The engineered strain BL21-OMT was inoculated into TB medium without methionine (control group) and with 1 g/L methionine supplementation (Met group), with the substrate concentration fixed at 150 mg/L naringenin. Sakuranetin concentration was determined using the same analytical method as described above.

## Result：

Sakuranetin production was compared between the control group (without methionine) and the Met group (with 1 g/L methionine supplementation). With methionine supplementation, sakuranetin production in the engineered strain BL21-OMT increased significantly from 17.16 ± 1.67 mg/L to 26.56 ± 0.80 mg/L, representing an average improvement of approximately 55%.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img9.webp)

Figure：Effect of 1 g/L methionine supplementation on sakuranetin production in BL21-OMT

## Conclusion：

Methionine supplementation significantly increased sakuranetin production in the BL21-OMT strain, which is likely due to enhanced intracellular SAM availability that promotes *F7-OMT*–mediated methylation. Nonetheless, the extent of improvement may be constrained by regulatory factors in the methionine–SAM cycle or by competing pathways consuming SAM.

# BL21-*OMT*-*YdaO*：Improved Sakuranetin production via *YdaO* co-expression

## Objective：

Building on the first-generation *F7-OMT* system, a second-generation engineered strain carrying the *ydaO* gene was constructed to strengthen the Met–SAM metabolic cycle, thereby enhancing the synthesis and recycling efficiency of the methyl donor SAM and further improving sakuranetin production. The ydaO element is an ATP-sensing riboswitch from *Bacillus subtilis* that binds ATP and dynamically regulates downstream gene transcription, thus enabling feedback control of cellular energy status.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img10.webp)

Figure: Role of the *YdaO* riboswitch in dynamic ATP homeostasis control.

## Method：

The *YdaO* coding gene was synthesized (BBa\_25Z8A0VX, Generalbiol, China) and codon-optimized for *E. coli,* with restriction sites incompatible with RFC#10 removed. The constitutive elements J23100-B0034 were used in cis, and the construct was cloned into the pSB1A3 vector via XbaI and SpeI restriction sites. In parallel, PCR and overlap extension were employed to place B0034-*YdaO* downstream of *F7-OMT*, generating a co-expression sequence of *F7-OMT* and *YdaO,* which was similarly cloned into pSB1A3 using XbaI and SpeI (Note:The *YdaO* operon that containing the *ydaO* riboswitch, vhb, and ptxD was introduced into the system. Hereafter, it is referred to simply as *YdaO*). As described above, recombinant plasmids were transformed into *E. coli* DH5α and BL21. Positive clones were screened on LB agar plates (supplemented with 100 μg/mL ampicillin and 1.5% agar) and verified by sequencing (Qingke, Beijing), resulting in the recombinant engineered strain BL21-*OMT*-*YdaO*. Strains were stored at –80 ℃ in 25% (v/v) glycerol as cryoprotectant. The culture conditions for the engineered strain were 37 ℃ and 150 rpm in LB broth (G3102, Servicebio, China) containing 100 μg/mL ampicillin for inoculation and expansion.

Subsequently, BL21-OMT and BL21-*OMT-YdaO* engineered strains were inoculated into TB medium containing 150 mg/L naringenin and fermented at 25 ℃ for 12 h. Sakuranetin concentration was measured using the same method as described above.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img11.webp)

Figure: Construction of BL21-*OMT*-*YdaO*. (A) The plasmid map of pSB1A3-*OMT-YdaO*. (B) Agarose gel electrophoresis of YadO (125 bp). We hypothesize that the fragment may have migrated out of the expected range due to excessively high voltage during electrophoresis.(C) The gene circuit of BL21-*OMT-YdaO.*

## Result：

This comparison evaluated sakuranetin production between the first-generation engineered strain BL21-*OMT* and the second-generation strain BL21-OMT-YdaO. The first-generation strain produced 18.96 ± 3.06 mg/L of sakuranetin, whereas the co-expression of *ydaO* in the second-generation strain significantly increased the yield to 36.10 ± 2.19 mg/L, representing an overall improvement of nearly 90%.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img12.webp)

Figure: Enhanced sakuranetin production in BL21-*OMT-YdaO* compared with BL21-*OMT*.

## Conclusion：

Co-expression of *ydaO* in the engineered strain BL21-OMT significantly enhanced sakuranetin production, nearly doubling the yield compared with the first-generation system. This demonstrates that strengthening the Met–SAM cycle through *ydaO*-mediated regulation is an effective strategy to improve methyl donor availability and optimize sakuranetin biosynthesis.

# BL21-*OMT-YdaO-CysE*: Further enhancement of Sakuranetin production by *CysE* co-expression

## Objective：

Although the introduction of *F7-OMT* and *ydaO* improved sakuranetin production, the yield still did not meet our expectations. Therefore, we aimed to develop a third-generation optimization system. Given that reproducing the entire design of the third-generation system would be overly complex, we focused on the most critical component, the CysE gene, whose circuit design has been described in the project overview. *CysE* regulates the cysteine → methionine metabolic pathway and thereby indirectly influences the synthesis of the methyl donor SAM, offering a potential route to further enhance sakuranetin production.

## Method：

To enhance methionine production in *E. coli*, the l-serine O-acetyltransferase coding gene CysE (BBa\_25MTY54W, Generalbiol, China) was synthesized and codon-optimized for *E. coli*, with restriction sites incompatible with RFC#10 removed. The constitutive elements J23100-B0034 were placed upstream of CysE as cis-acting regulatory elements. Using PCR and overlap extension, the J23100-B0034-CysE fragment was positioned downstream of *OMT-YdaO*, generating a co-expression sequence of OMT, *YdaO*, and CysE, which was cloned into the pSB1A3 vector via XbaI and SpeI restriction sites.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img13.webp)

Figure: Construction of BL21-*OMT-YdaO-C**ysE.* (A) The plasmid map of pSB1A3-*OMT-YdaO-C**ysE* (B) Agarose gel electrophoresis of *cysE* (819 bp).

As described above, the recombinant engineered strain BL21-*OMT-YdaO-CysE* was obtained. The strain was stored at –80 ℃ in 25% (v/v) glycerol as a cryoprotectant. The culture conditions for the engineered strain were 37 ℃ and 150 rpm. Inoculation and expansion were carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL ampicillin.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img14.webp)

Figure: The gene circuit of BL21-*OMT-YdaO-CysE(*From version 1 to version 3).

Subsequently, BL21-*OMT*, BL21-*OMT-YdaO* and BL21-*OMT-YdaO -CysE* were inoculated into TB medium containing 150 mg/L naringenin and fermented at 25 ℃ for 12 h. Sakuranetin concentration was measured using the same method as described above.

## Result：

With the optimization of the metabolic pathway, sakuranetin production was progressively enhanced. The first-generation BL21-OMT strain yielded 18.92 ± 2.58 mg/L; the second-generation BL21-*OMT-YdaO* system showed a significant increase to 31.07 ± 2.77 mg/L (p < 0.01); and the third-generation BL21-OMT-YdaO-CysEsystem further improved the yield to 45.21 ± 3.75 mg/L (p < 0.001), representing an increase of approximately 139% and 45% compared with the first- and second-generation systems, respectively.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img15.webp)

Figure: Comparison of sakuranetin production among different engineered strains (BL21-*OMT*, BL21-*OMT-YdaO*, and BL21-*OMT-YdaO-CysE*)

## Conclusion：

Introduction of the CysE gene in the third-generation system significantly enhanced endogenous methionine biosynthesis, thereby increasing the intracellular supply of SAM. As a result, the engineered strain achieved its highest sakuranetin production in the third-generation system, reaching 45.21 ± 3.75 mg/L. Co-expression of *F7-OMT*, *YdaO,* and *CysE* effectively enhanced sakuranetin production, providing a valuable strategy for the future optimization of sakuranetin biosynthetic pathways.

# Analysis of naringenin production

## Objective：

In the previous experiments, exogenous naringenin supplementation was required for sakuranetin production. To overcome this limitation, we aimed to engineer *E. coli* to endogenously produce naringenin, thereby reducing dependence on external substrate supply and supporting more efficient sakuranetin biosynthesis.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img16.webp)

Figure: Endogenous biosynthetic pathway for naringenin production.

## Method：

The coding genes TAL, 4CL, CHS, and CHI (BBa\_25886WFT, Generalbiol, China) were synthesized and codon-optimized for *E. coli*, with restriction sites EcoRI, XbaI, SpeI, and PstI removed to comply with RFC#10 standards. The constitutive promoter–RBS element J23100-B0034 was used to drive expression of TAL/4CL and CHS/CHI separately to avoid interference between dual promoters. A B0015 terminator sequence was added to both operons. The synthetic sequences were cloned into the pSB1A3 vector via XbaI and SpeI restriction sites. As described above, the recombinant engineered strain BL21-Nar was obtained. The strain was stored at –20 ℃ in 25% (v/v) glycerol as a cryoprotectant. Culture conditions for the engineered strain were 37 ℃ and 150 rpm. Inoculation and expansion were carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL ampicillin.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img17.webp)

Figure: Construction of BL21-Nar. (A) The plasmid map of pSB1A3-Nar. (B) Agarose gel electrophoresis of TAL (654 bp). (C) Agarose gel electrophoresis of 4CL (1710 bp). (D) Agarose gel electrophoresis of CHS (1167 bp). (E) Agarose gel electrophoresis of CHI (666 bp). （F）The gene circuit of BL21-Nar

A 100 μL aliquot of frozen BL21-Nar recombinant strain stock was inoculated into 5 mL LB medium supplemented with ampicillin (Amp+) and incubated overnight at 37 ℃, 150 rpm for activation. The overnight culture was transferred into 250 mL Erlenmeyer flasks containing 50 mL Amp+ LB medium, adjusted to an initial OD₆₀₀ of 0.1, and grown at 37 ℃, 180 rpm until OD₆₀₀ reached 1. Cells were harvested by centrifugation at 8000 × g for 5 min, then resuspended in M9 medium (A510881, Sangon Biotech) supplemented with 0.4% glucose (G6500, Innochem), 1 mM MgSO₄ (A40004, Innochem), 50 μM CaCl₂ (A01289, Innochem), 340 mg/L thiamine (BP892, Fisher), and 5 g/L CaCO₃ (A05990, Innochem). Cultures were incubated at 25 ℃, 180 rpm for 48 h.

For naringenin quantification, 1 mL of fermentation broth was mixed with 1 mL ethyl acetate, vortexed for 2 min, and centrifuged at 10,000 × g for 5 min. The upper organic phase was collected and dried overnight at 37 ℃. The residue was resuspended in 1 mL acetonitrile and analyzed at 290 nm using a microplate reader. Naringenin concentration was calculated from a standard curve (0–800 mg/L) prepared with authentic naringenin (A06896, Innochem).

For glucose quantification, 1 mL of fermentation broth was centrifuged at 10,000 × g for 5 min, and the supernatant was collected. Glucose levels were determined using a glucose assay kit (60408ES60, Yeasen, China). Samples were homogenized by ultrasonic disruption under ice-water conditions (power 300 W, 3–5 s per cycle, 30 s intervals, repeated 3–5 times) with 0.2–0.3 mL homogenization medium added. In a 96-well plate, 2.5 μL distilled water and 250 μL working solution were added to blank wells, 2.5 μL standard solution and 250 μL working solution to standard wells, and 2.5 μL sample with 250 μL working solution to sample wells. The plate was gently mixed and incubated at 37 ℃ for 10 min, after which absorbance was measured at 505 nm using a microplate reader. Glucose concentrations were calculated based on the standard curve.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img18.webp)

Figure: Principle of glucose assay based on the GOD–POD coupled colorimetric reaction

## Result：

Under fermentation conditions of 25 ℃ and 180 rpm, both naringenin concentration and glucose concentration were monitored over a 48 h period. In the recombinant strain BL21-Nar, naringenin production increased continuously from 0.0045 ± 0.0018 g/L at 0 h to 0.4214 ± 0.0474 g/L at 48 h. Meanwhile, glucose concentration decreased from an initial 39.88 ± 1.70 g/L to 15.48 ± 2.68 g/L, indicating a clear negative correlation between substrate consumption and product accumulation during the fermentation process.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img19.webp)

Figure: Time-course of naringenin production and glucose depletion during fermentation

## Conclusion：

In this study, a recombinant production strain capable of synthesizing naringenin was successfully constructed, demonstrating the feasibility of microbial naringenin biosynthesis. At present, this strain was developed as an independent system. Future work will focus on integrating it with the previously established sakuranetin production pathway, enabling a coupled system in which naringenin is both produced and simultaneously converted into sakuranetin.

# System 2： LPC Production System

We constructed an engineered strain expressing *B. ovatus*-derived PLA2 to promote the conversion of PC to LPC. Western blot analysis confirmed the successful expression of PLA2 in the engineered strain BL21-PLA2, with a distinct band observed at the expected molecular weight of PLA2 (≈30–42 kDa). ELISA results further showed that the control strain BL21 produced only 2.43 ± 1.28 μg/mL LPC, whereas the engineered strain BL21-PLA2 exhibited a significantly elevated LPC level of 74.23 ± 15.91 μg/mL. These findings demonstrate that overexpression of PLA2 was achieved at the protein level and effectively enhanced the strain’s capacity for substrate degradation and LPC biosynthesis, providing a novel strategy for targeting AD-related metabolic pathways.

# LPC Degradation Analysis

## Objective：

Building on the system in which sakuranetin alone served as the intervention molecule, we further explored whether synthetic biology could be leveraged to construct a multi-target intervention system, thereby providing more diverse and precise molecular tools for the prevention and treatment of AD. Mechanistic studies have shown that lysophosphatidylcholine (LPC) is a key metabolite generated from phosphatidylcholine (PC) through the catalytic action of phospholipase A2 (PLA2), and this pathway is particularly active in *Bacteroides ovatus*. Clinical data likewise indicate that both the abundance of *B. ovatus* in the gut and serum LPC levels are significantly reduced in AD patients compared with healthy controls, suggesting that the *B. ovatus*–PLA2–LPC metabolic axis plays an important role in AD pathogenesis.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img20.webp)

Figure: Microbiota - derived lysophosphatidylcholine alleviates Alzheimer's disease pathology via suppressing ferroptosis(Zha et al., 2025).

Based on these findings, we designed an innovative strategy: engineering *E. coli* BL21 to express the PLA2 gene derived from *B. ovatus* (BL21-PLA2). Leveraging the strain’s high cultivability and biosafety, this probiotic chassis is intended to efficiently catalyze the conversion of PC to LPC in the intestinal environment, thereby elevating LPC levels, mitigating Aβ deposition, and ultimately delaying AD progression.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img21.webp)

Figure：the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC)

## Method：

The phospholipase A2 (PLA2) coding gene (BBa\_25ST8FLD, Generalbiol, China) was synthesized and codon-optimized for *E. coli*, with restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI removed to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector. The gene was cloned into pET28a(m) via the *NdeI* and *XhoI* restriction sites. The recombinant plasmid was then transformed into *E. coli* DH5α. Positive clones were selected on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL kanamycin (Kana) and verified by sequencing (Qingke, Beijing), generating the recombinant strain DH5α-PLA2. Recombinant plasmids were extracted using a plasmid extraction kit (DP103, Tiangen) and transformed into *E. coli* BL21 (DE3) to obtain the engineered strain BL21-PLA2. The strain was stored at –80 ℃ in 25% (v/v) glycerol as a cryoprotectant. Culture conditions for the engineered strain were 37 ℃ and 150 rpm, with inoculation and expansion carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img22.webp)

Figure: Construction of BL21-PLA2. (A) The plasmid map of pET-28a(m)-PLA2. (B) Agarose gel electrophoresis of PLA2 (843 bp). (C) The gene circuit of BL21-PLA2.

The PLA2-overexpressing engineered strain was inoculated into 50 mL LB medium supplemented with Kana and cultured overnight. On the following day, the bacterial culture was harvested and centrifuged at 10,000 × g for 1 min. The cell pellet was resuspended in PBS and disrupted by ultrasonication (150 W, 1 s on/3 s off cycles, for a total of 20 min). The lysate was transferred into a fresh centrifuge tube and used as the intracellular protein sample. Protein solutions were mixed with 5× reducing sample buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 min. Proteins were separated on 15% SDS-PAGE gels at 120 V. Subsequently, proteins were transferred from the SDS-PAGE gel to a PVDF membrane under cold conditions. The membrane was blocked at room temperature with 5% non-fat milk in TBST for 1 h, followed by incubation at 4 ℃ overnight with a rabbit anti-PLA2 primary antibody (1:1000, e.g., Abcam or Cayman). After three washes with TBST (10 min each), the membrane was incubated at room temperature for 1 h with HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:1000 dilution, A0208, Beyotime). The membrane was then washed three times with TBST (10 min each). ECL reagents A and B were mixed at a 1:1 ratio and applied to the washed PVDF membrane placed on absorbent paper to remove excess liquid. The membrane was then covered with the prepared ECL solution for 1 min to ensure complete coverage. Excess liquid was removed with absorbent paper, and the membrane was exposed using a chemiluminescence imaging system under preset parameters.

The BL21-PLA2 and BL21 were inoculated at a 1:100 ratio into 5 mL LB medium containing 100 μg/mL kanamycin and cultured overnight at 37 ℃, 150 rpm for activation. Approximately 1 mL of the activated *E. coli* culture was then transferred into 50 mL M9 medium and adjusted to an initial OD600 of 0.1. The M9 medium contained 1× M9 salts (A510881, Sangon Biotech), 0.2% (w/v) glucose, 1 mM MgSO₄, and 50 μM CaCl₂, with kanamycin maintained at a final concentration of 100 μg/mL. When the culture reached OD₆₀₀ ≈ 0.6 at 37 ℃ and 150 rpm, 0.5 mM IPTG and 1 mg/mL egg yolk lecithin (L305002, Aladdin) were added, and incubation was continued for 24 h.

After fermentation, 1 mL of culture broth was centrifuged at 10,000 × g for 10 min, and the supernatant was collected and filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp). The filtrate was diluted 100-fold with PBS. The concentration of lysophosphatidylcholine (LPC) was then determined using an LPC ELISA kit based on a competitive binding assay. A standard curve was generated by plotting absorbance values against the log₁₀-transformed concentrations of LPC standards. Absorbance was measured at 450 nm using a microplate reader, and LPC concentrations in the samples were calculated according to the standard curve.

## Result：

Western blot analysis confirmed the expression of PLA2 in the engineered strain. A distinct band corresponding to the expected molecular weight of PLA2 (≈30-42 kDa) was detected in the PLA2-overexpressing strain. The intensity of the band indicated successful overexpression of PLA2, validating the efficiency of the expression system.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img23.webp)

Figure: Western blot analysis of PLA2 expression in engineered strain

A standard curve was established using LPC standard solutions at different concentrations, yielding the following fitted equation:

A450 = –0.915 × log₁₀(concentration) + 4.051, R² = 0.98.

The results demonstrated a strong linear relationship between A450 and log₁₀(concentration) with a high degree of fit (R² = 0.98), indicating that the assay is accurate and reproducible within the experimental concentration range. As LPC concentration increased, the A450 value decreased linearly, consistent with the principle of competitive ELISA: higher LPC levels in the sample result in fewer enzyme-labeled molecules bound to the antibody, weaker color development, and thus lower A450 readings.

Comparative analysis further showed that the unmodified BL21 control strain produced only a very low level of LPC (2.43 ± 1.28 μg/mL), whereas the recombinant strain BL21-PLA2 exhibited a markedly higher LPC level of 74.23 ± 15.91 μg/mL (p < 0.01). These findings indicate that overexpression of the PLA2 gene greatly enhanced the engineered strain’s capacity for substrate degradation and LPC generation.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img24.webp)

Figure: LPC detection and analysis of engineered strains. (A) LPC standard calibration curve; (B) Comparison of LPC production (μg/mL) between control strain BL21 and engineered strain BL21-PLA2.

## Conclusion：

Western blot analysis confirmed successful expression of PLA2 in the engineered strain BL21-PLA2, which showed markedly increased LPC production in vitro compared with the control strain. These findings validate the feasibility of the *B. ovatus*–PLA2–LPC metabolic pathway and provide an engineering basis for enhancing LPC levels in the gut. Combined with our previous optimization of sakuranetin biosynthesis, this multi-target strategy offers a new approach for intervening in the progression of Alzheimer’s disease.

# System 3： Safety Suicide System

To improve biosafety, we designed a dual-layer suicide system with both artificial control and environmental triggering. In the pBAD–T4 lysis module, arabinose addition induced cell lysis within 4 h, and cell density dropped to 0.19 ± 0.16 at 20 h, showing almost no growth. In the pCspA–T4 lysis module, cell growth was strongly inhibited at 16 ℃, with OD600 reaching only 0.34 ± 0.08 at 8 h versus 0.83 ± 0.06 in the control. Together, these results show that the system allows artificial clearance when needed and automatic lysis at low temperature, effectively reducing the risk of engineered strain spread.

# DH5α-pBAD-mRFP：Validation of pBAD promoter activity for inducible suicide system

## Objective：

To verify the effectiveness of the arabinose-inducible promoter pBAD, we linked mRFP downstream as a reporter gene and tested its induction under different L-arabinose concentrations. The experiment aimed to assess whether pBAD can achieve controllable and dose-dependent gene expression, thereby providing evidence for the reliability of the pBAD–T4 lysis suicide system.

## Method：

The arabinose-inducible promoter pBAD (K808000), RBS B0034, and the red fluorescent protein mRFP were synthesized. The mRFP gene was placed downstream of pBAD-B0034. The mRFP sequence was codon-optimized for *E. coli* and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The construct pBAD-mRFP was cloned into the pSB1A3 vector between the *XbaI* and *SpeI* restriction sites. The recombinant plasmid was transformed into *E. coli* DH5α, and positive clones were screened on LB agar plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing), yielding the engineered strain. The engineered strain was cultured at 37 ℃ in LB broth for inoculation and expansion. Bacterial growth was monitored using a spectrophotometer (GenStar, China) by measuring optical density at 600 nm (OD600).

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img25.webp)

Figure: Construction of DH5α-pBAD-mRFP. (A) The plasmid map of pSB1A3-pBAD-mRFP. (B) Agarose gel electrophoresis of araC-pBAD (1210 bp). Since the pBAD fragment is relatively short, it was linked with the araC regulatory sequence. (C) The gene circuit of DH5α-pBAD-mRFP.

The arabinose-inducible reporter strain was inoculated at a 1:100 ratio into 5 mL LB medium containing 50 μg/mL ampicillin, with different concentrations of L-arabinose added. Cultures were incubated at 37 ℃ and 180 rpm in a shaking incubator. At designated time points, 200 μL of culture was sampled. Fluorescence intensity (excitation: 584 nm; emission: 607 nm) and OD600 were measured using a microplate reader (Multiskan GO). Normalized fluorescence was calculated as the ratio of fluorescence to OD600（Fluorescence/OD600）.

## Result：

The experimental results were expressed as normalized fluorescence (Fluorescence/OD600) in arbitrary units (AU). The data showed that the activity of the pBAD promoter exhibited a clear dose-dependent response to exogenous L-arabinose concentration.

Under 0% arabinose, fluorescence remained at a low background level across all time points (0 h: 11.19 ± 0.56 AU; 2 h: 7.30 ± 0.52 AU; 4 h: 11.06 ± 0.63 AU; 8 h: 9.90 ± 1.98 AU), indicating that the pBAD promoter was inactive.

At 0.05% arabinose, fluorescence increased rapidly to 69.80 ± 5.25 AU at 2 h, reached 274.62 ± 77.33 AU at 4 h, and further rose to 451.47 ± 139.93 AU at 8 h, suggesting that even a low concentration could trigger significant induction.

At 0.2% arabinose, induction was more pronounced, with fluorescence reaching 524.11 ± 56.77 AU at 4 h and 829.13 ± 62.16 AU at 8 h.

At 0.5% arabinose, the induction was strongest, with fluorescence rising to 630.96 ± 44.09 AU at 4 h and exceeding 1089.19 ± 108.40 AU at 8 h, demonstrating the strong driving capacity of pBAD.

In summary, these results clearly indicate that the expression strength of the pBAD promoter increases with rising arabinose concentrations, showing rapid response and good reproducibility.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img26.webp)

Figure: Dose- and time-dependent induction of pBAD promoter activity by L-arabinose

## Conclusion：

This experiment confirmed the dose-dependent and strong inducible properties of the pBAD promoter. It maintained low basal expression in the absence of arabinose, while rapidly and robustly driving downstream gene expression upon arabinose supplementation.

# DH5α-pBAD–T4 lysis: Arabinose-inducible suicide system

## Objective：

To enhance the biosafety of engineered strains, we designed an arabinose-inducible suicide system (pBAD–T4 lysis). In this system, T4 holin and T4 lysozyme were placed downstream of the pBAD promoter, enabling engineered bacteria to undergo self-lysis in the presence of exogenous L-arabinose, thereby allowing artificially controlled clearance when needed(Couse, 1968).

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img27.webp)

Figure: Design principle of the arabinose-inducible suicide system (replacement of mRFP with T4-lysis downstream of pBAD)

## Method：

The T4 holin (BBa\_254YB8C8) and T4 lysozyme (BBa\_25L8X41M) (T4 lysis) coding genes were inserted downstream of pBAD-B0034. The T4 lysis sequence was codon-optimized for *E. coli* and restriction sites EcoRI, XbaI, SpeI, and PstI were removed to comply with RFC#10 standards (Genewiz, USA). The pBAD-B0034–T4 lysis construct was cloned into the pSB1A3 plasmid at the XbaI and SpeI restriction sites and subsequently transformed into *E. coli DH5α*.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img28.webp)

Figure: Construction of DH5α-pBAD-T4 lysis. (A) The plasmid map of pSB1A3-pBAD-T4 lysis. (B) Agarose gel electrophoresis of T4 Holin (657 bp). (C) Agarose gel electrophoresis of T4 Lysozyme (492 bp). (D) The gene circuit of DH5α-pBAD-T4 lysis.

The arabinose-inducible suicide strain was cultured overnight, and 100 μL of the culture was inoculated into 5 mL LB medium containing 50 μg/mL ampicillin. After 4 h of cultivation, 0.5 mM L-arabinose was added, and the culture was incubated at 37 ℃ with shaking at 180 rpm. At different time points, 200 μL samples were taken to measure the OD600 of the cells.

## Result：

The OD600 changes of the control group (DH5α) and the experimental group (DH5α/pBAD-T4 lysis) were as follows: at 0 h, both strains had similar initial densities of 0.36 ± 0.11 and 0.38 ± 0.08, respectively. At 2 h, the OD600 values increased to 0.67 ± 0.08 and 0.65 ± 0.11, both showing normal exponential growth. After the addition of L-arabinose at 4 h, the control group continued to grow to 1.11 ± 0.17, while the experimental group reached 0.86 ± 0.12, with no significant difference observed. Subsequently, at 8 h, the OD600 of the control group rose to 1.60 ± 0.15, whereas the experimental group dropped sharply to 0.29 ± 0.16. By 20 h, the control group further increased to 2.31 ± 0.22, while the experimental group remained at only 0.19 ± 0.16, indicating almost no growth. These results demonstrate that after induction at 4 h, the pBAD-T4 lysis engineered strain underwent significant cell lysis in response to L-arabinose, whereas the control strain maintained normal growth.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img29.webp)

Figure：Growth dynamics of DH5α and pBAD–T4 lysis strains after L-arabinose induction at 4 h

## Conclusion：

This study confirmed the effectiveness of the arabinose-inducible suicide system (pBAD–T4 lysis). Without induction, engineered and control strains grew normally. After L-arabinose addition at 4 h, the engineered strain underwent rapid self-lysis, with growth almost completely lost by 8–20 h. These results demonstrate that the pBAD–T4 lysis module functions as a reliable, controllable safety switch. Importantly, it allows the clearance of residual engineered bacteria *in vivo* through oral administration of L-arabinose, thereby reducing potential environmental and biosafety risks.

# DH5α-pCspA–mRFP: Validation of cold-inducible reporter activity

## Objective：

In the previous experiment, we confirmed the controllability of the arabinose-inducible pBAD system and demonstrated its artificial killing function through the pBAD–T4 lysis module. However, artificial induction alone is not sufficient to ensure biosafety. Therefore, in this experiment, we tested the cold-inducible promoter pCspA at different temperatures to evaluate its expression activity and verify its potential as an environment-triggered safety switch for a cold-inducible suicide system.

## Method：

The cold-inducible promoter pCspA (K4987003) was synthesized. The mRFP gene was placed downstream of pCspA. The mRFP sequence was codon-optimized for *E. coli*, and the EcoRI, XbaI, SpeI, and PstI restriction sites were removed to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector, the pCspA-mRFP construct was cloned between the XbaI and SpeI restriction sites. The recombinant plasmid was transformed into *E. coli* DH5α, and positive clones were screened on LB (Luria Bertani) agar plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing), generating the engineered strain. The engineered strain was cultured at 37 ℃ in LB broth for inoculation and amplification. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a spectrophotometer (GenStar, China).

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img30.webp)

Figure: Construction of DH5α-pCspA-mRFP. (A) The plasmid map of pSB1A3-pCspA-mRFP. (B) Agarose gel electrophoresis of pCspA-mRFP (738 bp); since the pCspA promoter is too short, the mRFP reporter was placed downstream for validation. (C)The gene circuit of DH5α-pCspA-mRFP.

The overnight-activated cold-inducible reporter strain was inoculated into LB medium at a 1:100 ratio and cultured at different temperatures for 12 h under shaking conditions (180 rpm). A 200 μL aliquot of culture was collected, and fluorescence (excitation 584 nm, emission 607 nm) together with OD600 was measured using a microplate reader (Thermo Fisher Scientific, USA). The normalized fluorescence ratio (Fluorescence/OD600) was then calculated.

## Result：

Under low-temperature conditions, the pCspA promoter strongly drove fluorescent expression. At 16 ℃, fluorescence rapidly increased from 32.77 ± 14.58 AU at 2 h to 168.20 ± 20.80 AU at 6 h, reaching 270.84 ± 19.36 AU at 12 h. At 25 ℃, induction was moderate, with fluorescence reaching 240.04 ± 14.51 AU at 12 h, slightly lower than at 16 ℃. In contrast, at 37 ℃, fluorescence remained at a low background level, reaching only 67.92 ± 22.24 AU at 12 h. These results clearly demonstrate that pCspA activity is significantly enhanced at lower temperatures, enabling strong induction under cold conditions while maintaining minimal basal expression at higher temperatures.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img31.webp)

Figure: Temperature-dependent activation of pCspA promoter driving mRFP expression

## Conclusion：

The cold-inducible promoter pCspA can strongly drive downstream gene expression under low-temperature conditions while maintaining minimal activity at 37 ℃. This “on at low temperature, off at high temperature” feature makes it an ideal environmental sensor, capable of triggering suicide modules when engineered bacteria accidentally enter external low-temperature environments to prevent dissemination, while remaining inactive in the gut (37 ℃) to preserve normal function. This provides a solid foundation for subsequent replacement of mRFP with T4 lysis.

# DH5α-pCspA–T4 lysis: Validation of cold-inducible suicide system

## Objective：

We previously confirmed that the cold-inducible promoter pCspA can effectively drive gene expression at low temperatures. To further assess its biosafety potential, we constructed the pCspA–T4 lysis suicide system and tested the growth of engineered strains at 16 ℃ to determine whether the system could trigger cell lysis under low-temperature conditions and achieve automatic clearance.

![Objective： Diagram](https://static.igem.wiki/teams/6029/results/img32.webp)

Figure: Design principle of the cold-inducible suicide system (replacement of mRFP with T4-lysis downstream of pCspA)

## Method：

The T4 holin and T4 lysozyme (T4 lysis) genes were placed downstream of the pCspA promoter, codon-optimized, and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards. The recombinant fragment was cloned into the pSB1A3 plasmid and transformed into *E. coli* DH5α, generating the pCspA–T4 lysis engineered strain.

![Method： Diagram](https://static.igem.wiki/teams/6029/results/img33.webp)

Figure: The plasmid map of pSB1A3-pCspA-T4 lysis.

The cold-inducible suicide strain and control strain were cultured overnight, and 100 μL of each was inoculated into 5 mL of LB medium supplemented with ampicillin (50 μg/mL). Cultures were incubated at 16 ℃ with shaking at 180 rpm. At designated time points, 200 μL samples were collected to measure bacterial OD600.

## Result：

The OD600 changes of the control strain (DH5α) and the experimental strain (DH5α/pCspA–T4 lysis) were as follows : at 0 h, values were 0.21 ± 0.03 and 0.19 ± 0.05, respectively, with no significant difference between the two groups. At 2 h, the OD600 of the control strain increased to 0.36 ± 0.04, while the experimental strain remained at 0.22 ± 0.06. At 4 h, the control strain further increased to 0.60 ± 0.07, whereas the experimental strain reached only 0.31 ± 0.06. By 8 h, the control strain grew to 0.83 ± 0.06, while the experimental strain remained at 0.34 ± 0.08. These results indicate that under low-temperature conditions, the growth of the pCspA–T4 lysis engineered strain was significantly inhibited, with OD600 consistently maintained at low levels, while the control strain proliferated normally.

![Result： Diagram](https://static.igem.wiki/teams/6029/results/img34.webp)

Figure：Growth inhibition of DH5α/pCspA–T4 lysis at 16 ℃ compared with wild-type DH5α

## Conclusion：

The cold-inducible suicide system pCspA–T4 lysis effectively restricted bacterial growth at low temperatures, confirming its ability to trigger efficient self-lysis. Together with the arabinose-inducible pBAD–T4 lysis system, they form a complementary “dual-safeguard” design. The first layer, pCspA–T4 lysis, is activated by environmental signals and triggers cell lysis when engineered bacteria are exposed to low temperatures outside the host, preventing their survival and spread. The second layer, pBAD–T4 lysis, can be activated by adding arabinose, enabling researchers or users to deliberately eliminate engineered strains inside the host or during experiments. This combined strategy of “environment-triggered + manually controlled” ensures both stable metabolic function within the host and rapid clearance when needed, greatly reducing biosafety risks.

# Reference

[1] Sun, Q., Gao, S., Yu, S., Zheng, P., & Zhou, J. (2022). Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance. Synthetic and systems biotechnology, 7(4), 1117-1125.

[2] Zha, X., Liu, X., Wei, M., Huang, H., Cao, J., Liu, S., ... & Zhang, C. (2025). Microbiota-derived lysophosphatidylcholine alleviates Alzheimer’s disease pathology via suppressing ferroptosis. Cell metabolism, 37(1), 169-186.

[3] Couse, N. L. (1968). Control of lysis of T4-infected Escherichia coli. Journal of Virology, 2(3), 198-207.

[4] <https://registry.igem.org/parts/bba-k808000>

[5] [https://parts.igem.org/Part:BBa\_K4987003](https://parts.igem.org/Part%3ABBa_K4987003)

{% endblock %}