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.
| 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 |
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.
| 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 3 focused on developing a dual-layer safety suicide system in E. coli to minimize biosafety risks. First, the arabinose-inducible promoter
| 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. |
