iGEM 2025 — Team csmu-taiwan

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{% 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 %}
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![](https://static.igem.wiki/teams/5898/header/notebook-header.webp)

* 08 May – 14 May (Week 19)

  This week we performed the first attempts at transformation and colony growth.

  + On **May 8**, LB agar plates were prepared, and plasmids were transformed into E. coli using heat shock. Colonies were left to grow while replicating the plasmids.
  + On **May 12**, we prepared 5 mL TB medium supplemented with 5 μL antibiotics(ampicillin) in four tubes. Four single colonies were inoculated. Tubes were incubated overnight.
  + On **May 13**, no growth was observed from the inoculated cultures. Colonies were re-picked and grown again.
  + On **May 14**, after 14h incubation, no signs of bacterial growth were observed. Possible reasons included excessive antibiotic concentration or the culture medium being unsuitable. Switching to LB medium was suggested for future attempts.
* 19 May – 22 May (Week 20)

  This week we restarted transformations and checked plasmid yields.

  + On **May 19**, to avoid troubleshooting variable by variable, we repeated the transformation with E.coli DH5α. Two selective conditions were used: Erythromycin and Ampicillin plates. Cultures were grown ~18 h.
  + On **May 20**, colonies were picked: five tubes from Erythromycin plates and three from Ampicillin plates were inoculated into 5 mL culture medium and incubated at 37 °C.
  + On **May 21–22**, most cultures grew well. For ermGFP plasmid, all five cultures showed growth. For mRFP1 plasmid, only one out of three tubes had stronger growth. Plasmid extraction resulted in low concentrations (30–50 ng/µL). After re-elution, concentrations improved to ~80 ng/µL in some samples, but still insufficient overall.
  + Bad news: the plasmid supplier reported they could not construct plasmids with Erythromycin selection.
* 26 May – 27 May (Week 21)

  This week we focused on mRFP1 plasmid growth and plasmid extraction.

  + On **May 26**, six cultures of pLEM415-IdhL-mRFP1 were prepared (3 in TB medium, 3 in LB medium).
  + On **May 27**, two LB cultures showed poor growth. Plasmid extraction was performed on the remaining cultures. Colonies in some tubes displayed visible pink coloration, indicating expression of red fluorescent protein. The plasmid was confirmed to be a high-copy plasmid. NanoDrop results showed good concentration, but the purity ratios were inconsistent. After discussion, two plasmids were selected for submission to the company.

    Table 1. Nanodrop concentration for pLEM415-IdhL-mRFP1 plasmids.

    | sample name | ng/µL | A260/A280 | A260/A230 | A260 | A280 |
    | --- | --- | --- | --- | --- | --- |
    | sample 1 | 570.5 | 2.08 | 2.38 | 11.41 | 5.47 |
    | sample 2 | 383.5 | 2.18 | 2.23 | 7.67 | 3.52 |
    | sample 3 | 595.5 | 2.00 | 2.40 | 11.91 | 5.95 |
    | sample 4 | 637.9 | 2.07 | 2.41 | 12.76 | 6.17 |
  + A cloning strategy discussion was held:
    - The original plasmid contained the insert: IdhL-FLAG-mRFP1, flanked by restriction sites ApaI (GGGCCC) and ClaI (ATCGAT).
    - Plan: excise the FLAG-mRFP1 fragment and replace it with the designed sequence (IdhL–[our sequence]).
* 15 July (Week 27)

  Deletions and mutations were detected in some constructs, requiring a redesign of the cloning strategy.
* 06 August (Week 32)

  We designed three prototype constructs of anti-IL-23 single-chain variable fragments (scFvs). In addition to these novel prototypes, we also modified the variable regions of two clinically approved IL-23-targeting antibodies into scFv format: **Risankizumab**, which neutralizes the **p19 subunit** of IL-23 (hereafter referred to as **p19** in this notebook), and **Ustekinumab**, which targets the **p40 subunit** (hereafter referred to as **p40** in this notebook). All three prototype scFvs, along with the scFv versions of Risankizumab and Ustekinumab, were submitted to a biotechnology company for gene synthesis and recombinant expression.
* 11 Aug – 19 Aug (Week 33)

  This week we focused on plasmid amplification in E.coli DH5α, plasmid extraction, quality check, and subsequent transfection into HEK293T cells.

  + On **Aug 11**, DH5α cultures harboring the CMV plasmid were inoculated in LB medium and incubated overnight at 37 °C.
  + On **Aug 12**, plasmid extraction was performed from the overnight cultures. NanoDrop measurements were taken to determine plasmid concentration and purity.

    Table 2. Nanodrop concentration for CMV plasmids containing ScFv-IL23 variants (prototype A-C), positive control (PC-p19, and PC-p40).

    | Insertion (sample name) | Replicate | ng/µL | A260/A280 | A260/A230 | A260 | A280 |
    | --- | --- | --- | --- | --- | --- | --- |
    | ScFv IL23 prototype A | 1-1 | 507.2 | 1.88 | 2.28 | 10.14 | 5.41 |
    | ScFv IL23 prototype A | 1-2 | 375.0 | 1.87 | 2.23 | 7.50 | 5.41 |
    | ScFv IL23 prototype B | 2-1 | 427.2 | 1.87 | 2.26 | 8.54 | 4.02 |
    | ScFv IL23 prototype B | 2-2 | 536.8 | 1.88 | 2.29 | 10.74 | 4.58 |
    | ScFv IL23 prototype C | 3-1 | 464.8 | 1.87 | 2.26 | 9.30 | 5.71 |
    | ScFv IL23 prototype C | 3-2 | 527.1 | 1.87 | 2.27 | 10.54 | 4.97 |
    | PC-p19 | 4-1 | 279.7 | 1.86 | 2.20 | 5.59 | 3.01 |
    | PC-p19 | 4-2 | 295.9 | 1.86 | 2.22 | 5.92 | 3.18 |
    | PC-p40 | 5-1 | 425.4 | 1.87 | 2.28 | 8.51 | 4.55 |
    | PC-p40 | 5-2 | 491.1 | 1.87 | 2.26 | 9.82 | 5.26 |

    ps. Each insertion construct was cultured and plasmid DNA extracted in duplicate (labeled -1 and -2). For each pair, the replicate with higher yield/purity was highlighted and selected for downstream use.
  + On **Aug 14-15**, additional plasmid preparations were carried out to obtain sufficient DNA for transfection. Gel electrophoresis confirmed plasmid integrity.
  + On **Aug 16-17**, HEK293T cells were maintained in log-phase growth and seeded at appropriate density for transfection.
  + On **Aug 19**, transfection was performed in HEK293T cells using purified plasmid DNA together with TransIT-X2 transfection reagent, according to the manufacturer’s instructions. The cells were returned to the incubator (37 °C, 5% CO₂) for expression studies.
* 20 Aug – 23 Aug (Week 34)

  This week, we focused on testing IL-23 antibody secretion from transfected HEK293T cells. Samples from both culture supernatants and cell lysates were collected and analyzed by SDS-PAGE followed by Western blotting to evaluate expression and secretion efficiency.

  + On **Aug 20,** culture supernatant and corresponding cell pellets were collected from transfected HEK293T cells.
  + On **Aug 21**, cell lysis was performed to extract intracellular proteins. Both supernatant and lysate fractions were stored at –20 °C for downstream analysis.
  + On **Aug 22**, protein concentration was quantified using BCA assay to normalize sample loading.

  Table 3. BCA Assay Protein Quantification Results for NCV and anti-IL23 Samples.

  | Sample Name | OD562 | Conc. (μg/μL) |
  | --- | --- | --- |
  | NCV | 0.349 | 7.992 |
  | anti-IL23 scFv 1-1 | 0.347 | 7.935 |
  | anti-IL23 scFv 2-2 | 0.338 | 7.677 |
  | anti-IL23 scFv 3-2 | 0.340 | 7.735 |
  | anti-IL23 scFv p19 | 0.329 | 7.420 |
  | anti-IL23 scFv p40 | 0.355 | 8.164 |

  + On **Aug 23**, SDS-PAGE was performed to compare protein content in cell lysate.
* 25 Aug – 29 Aug (Week 35)

  This week we carried out Western blot analysis to detect anti-IL23 expression in transfected HEK293T cells.

  + On **Aug 25**, Proteins from SDS-PAGE gels were transferred onto PVDF membranes. Membranes were blocked with blocking buffer (room temperature) and then incubated overnight at 4 °C with primary antibodies (1° Ab):
    - **Anti-His tag (mouse)** → for detecting His-tagged proteins.
    - **Anti-pSTAT3 (Y705, rabbit)** → for detecting phosphorylated STAT3 at tyrosine 705.
  + On **Aug 26**, After washing, membranes were incubated with secondary antibodies (2° Ab):
    - **Anti-mouse HRP** → detecting His-tag primary antibody.
    - **Anti-rabbit HRP** → detecting pSTAT3 primary antibody.

    HRP conjugates enabled chemiluminescent detection of target proteins.
  + On **Aug 27**,we performed a second Western blot targeting **STAT3** and **GAPDH** to validate downstream signaling effects and assess protein loading, respectively. Additionally, to prepare for downstream transfection or stimulation assays, we also replaced the culture medium of previously seeded HEK293T cells with Opti-MEM (serum-reduced medium), allowing better compatibility with transfection reagents and minimizing serum interference.
    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-1.avif)

    **Figure 1. Validation of scFv expression in both cell lysate and culture supernatant collected on Aug 21.**
  + On **Aug 29**, we collected supernatant and lysate of HEK239T. Samples were concentrated and prepared for Western blotting.

    **Table 4. Cell lysate samples (target 110 µg/eppendorf)**

    | Sample | Conc. (µg/µL) | Lysate (µL) | NETN (µL) | 6× dye (µL) |
    | --- | --- | --- | --- | --- |
    | NCV | 11.1133 | 9.90 | 56.1 | 17 |
    | anti-IL-23 1-1 | 10.96552 | 10.03 | 56.0 | 17 |
    | anti-IL-23 2-2 | 12.0000 | 9.17 | 56.8 | 17 |
    | anti-IL-23 3-2 | 11.01478 | 9.99 | 56.0 | 17 |
    | anti-IL-23 4-2 | 12.54187 | 8.77 | 57.2 | 17 |
    | anti-IL-23 5-2 | 10.96552 | 10.03 | 56.0 | 17 |

    ---

    **Table 5. Concentrated supernatant samples (target 35 µg/eppendorf)**

    | Sample | Conc. (µg/µL) | Sup. (µL) | NETN (µL) | 6× dye (µL) |
    | --- | --- | --- | --- | --- |
    | NCV | 3.625616 | 9.65 | 18.35 | 7 |
    | anti-IL-23 1-1 | 3.773399 | 9.28 | 18.72 | 7 |
    | anti-IL-23 2-2 | 4.26601 | 8.20 | 19.80 | 7 |
    | anti-IL-23 3-2 | 3.625616 | 9.65 | 18.35 | 7 |
    | anti-IL-23 4-2 | 4.70936 | 7.43 | 20.57 | 7 |
    | anti-IL-23 5-2 | 3.330049 | 10.51 | 17.49 | 7 |
* Sep 1 – Sep 7 (Week 36)

  This week we focus on the continuation and functional verification for our scFv constructs. We completed Western blot analysis for our samples, and HEK293T cells were subcultured and transfected with additional constructs, followed by supernatant and lysate collection for further protein analysis.

  + On **Sep 1**, we ran the SDS-PAGE for the five samples prepared on Aug 29, which included five anti-IL-23 constructs.
  + On **Sep 2**, ELISA plates were coated with 5ng IL23 to evaluate whether our anti-IL-23 scFv could bind to IL-23 subunits. After coating, ELISA was performed to test antibody-antigen interaction. In parallel, we developed Western blot membranes from the August 29 SDS-PAGE. The blot confirmed whether the transfected cells had successfully secreted scFv antibodies into the supernatant.
    ![](https://static.igem.wiki/teams/5898/notebook/notebook-f2.avif)

    **Figure 2. Validation of scFv expression in cell lysate collected on Aug 29**

    ![](https://static.igem.wiki/teams/5898/notebook/notebook-figure3.webp)

    **Figure 3.Validation of scFv expression in culture supernatant collected on August 29, compared with cell lysate samples in lanes 1 and 2.**
  + On **Sep 3**, we performed ELISA detection for plates coated on September 2. After blocking, we added primary antibodies targeting IL-23 and tag sequences, followed by HRP-conjugated secondary antibodies and TMB substrate for color development. Absorbance was measured to evaluate binding of scFv p19 and p40 to IL-23 subunits. Consistently higher OD450 values were observed in column 2 (p40), indicating stronger or more accessible antibody binding. However, inconsistent blank values (H1 = 0.279, H2 = 0.068) suggest variation in background signal, and data interpretation should be made with caution.

    **Table 6. ELISA OD450 values validation of the binding capability of anti-IL-23 scFv p19 and p40 collected on Aug 29.**

    | Supernatant Protein Amount (μg) | p19 | p40 |
    | --- | --- | --- |
    | 0 | 0.279 | 0.068 |
    | 0.3125 | 0.163 | 0.377 |
    | 0.625 | 0.241 | 0.479 |
    | 1.25 | 0.237 | 0.519 |
    | 2.5 | 0.291 | 0.620 |
    | 5 | 0.473 | 0.613 |
    | 10 | 0.454 | 0.590 |
    | 20 | 0.658 | 0.591 |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-4.avif)

    **Figure 4. ELISA OD450 curves validation of the binding capability of anti-IL-23 scFv p19 and p40 collected on Aug 29.**

    **Table 7. BCA assay of total protein concentration in cell lysates.**

    | Sample name | OD562 | conc. (µg/µL) |
    | --- | --- | --- |
    | NCV | 0.279 | 11.11 |
    | anti-IL23 1-1 | 0.276 | 10.97 |
    | anti-IL23 2-2 | 0.297 | 12.00 |
    | anti-IL23 3-2 | 0.277 | 11.01 |
    | PC-p19 4-2 | 0.308 | 12.54 |
    | PC-p40 5-2 | 0.276 | 10.97 |

    **Table 8. BCA assay of total protein concentration in culture supernatant.**

    | Sample name | OD562 | Conc. (µg/µL) |
    | --- | --- | --- |
    | NCV | 0.127 | 3.63 |
    | anti-IL23 1-1 | 0.130 | 3.77 |
    | anti-IL23 2-2 | 0.140 | 4.27 |
    | anti-IL23 3-2 | 0.127 | 3.63 |
    | PC-p19 4-2 | 0.149 | 4.71 |
    | PC-p40 5-2 | 0.121 | 3.33 |
  + On **Sep 4**, HEK293T cells were subcultured to expand stocks and prepare conditioned medium for subsequent treatment of Caco‑2 cells. In parallel, ELISA plates were coated with 5ng IL23 to valid the binding capability of anti‑IL‑23 scFv 1‑1, p19 and p40 collected from culture supernatant, then incubated at 4 °C overnight for the next day’s detection.
  + On **Sep 5**, we performed ELISA detection for plates coated on September 4. After blocking, we added primary antibodies targeting IL-23 and tag sequences, followed by HRP-conjugated secondary antibodies and TMB substrate for color development. Absorbance was measured to evaluate binding of scFv 1-1, p19 and p40 to IL-23 subunits.

    The signals do not decrease monotonically down the columns as expected for a serial dilution. This indicates a technical issue rather than true binding behavior. Because the concentration trend is inconsistent, these OD values are not reliable for quantitative comparison. We will repeat the assay with the corrected workflow and document blank‑subtracted, monotonic standard curves before interpreting sample binding.

    In parallel, we transfected HEK293T cells with new constructs, which will later be used to treat Caco-2 cells in downstream functional assays.

    **Table 9. ELISA OD450 values for validating the binding capability of scFvs collected from culture supernatants on Aug 29.**

    | Supernatant Protein Amount (μg) | anti-IL23 scFv p19 sup | anti-IL23 scFv p40 sup | anti-IL23 scFv 1-1 sup |
    | --- | --- | --- | --- |
    | 20 | 0.835 | 0.708 | 0.725 |
    | 10 | 0.464 | 0.681 | 0.545 |
    | 5 | 0.487 | 0.742 | 0.715 |
    | 2.5 | 0.463 | 0.853 | 0.314 |
    | 1.25 | 0.704 | 0.676 | 0.109 |
    | 0.625 | 0.285 | 1.230 | 0.386 |
    | 0.3125 | 0.260 | 1.102 | 0.118 |
    | 0 | 0.113 | 0.778 | 0.105 |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-5.avif)

    **Figure 5.ELISA for validating the binding capability of scFvs collected from culture supernatants on Aug 29.**
  + On **Sep 6**, we replaced the culture medium of HEK293T cells transfected on September 5. The transfection was intended to produce scFv antibodies for subsequent Caco-2 treatment and analysis.The old medium was carefully aspirated, and replaced with serum-reduced medium (Opti-MEM) to support continued protein expression.Cells were incubated at 37 °C, 5% CO₂ for a total of 72 hours, allowing sufficient time for antibody secretion into the supernatant, which will be harvested for downstream Western blot and ELISA assays.
* Sep 8 – Sep 14 (Week 37)

  This week focused on downstream functional assays for anti-IL-23 scFv constructs. We harvested supernatants and lysates from transfected HEK293T cells and continued protein detection via Western blot. In parallel, E. coli DH5α was cultured for plasmid amplification. On September 12, we treated Caco-2 cells with IL-23 and scFv samples to evaluate downstream signaling. We then collected cells for Western blot analysis of pSTAT3 and JAK expression. ELISA coating and detection were also carried out to monitor binding activity.

  + On **Sep 8**, supernatants were harvested from HEK293T cells transfected with five different anti-IL-23 scFv constructs (#1–p40), as well as a negative control vector (NCV). All samples were rapidly frozen in liquid nitrogen and stored for future use.
    A BCA assay was performed to quantify total protein concentrations.Samples were diluted and mixed with loading buffer according to calculated volumes to normalize total protein input to 15 µg per lane for SDS-PAGE. A new gel was cast for downstream Western blot analysis.

    **Table 10. BCA assay and sample loading volumes for concentrated HEK293T supernatants (target 15 µg antibody per lane in WB)**

    | Sample | OD562 | Conc. (µg/µL) | Volume of sample (µL) | PBS (µg) | 6× dye (µL) |
    | --- | --- | --- | --- | --- | --- |
    | NCV | 0.118 | 1.78 | 8.43 | 3.57 | 3 |
    | anti-IL23 scFv 1 | 0.126 | 2.06 | 7.27 | 4.73 | 3 |
    | anti-IL23 scFv 2 | 0.114 | 1.64 | 9.16 | 2.84 | 3 |
    | anti-IL23 scFv 3 | 0.125 | 2.03 | 7.39 | 4.61 | 3 |
    | anti-IL23 scFv p19 | 0.132 | 2.28 | 6.59 | 5.41 | 3 |
    | anti-IL23 scFv p40 | 0.123 | 1.96 | 7.66 | 4.34 | 3 |

    **Table 11. BCA assay for cell lysates.**

    | Sample | OD562 | Conc. (µg/µL) |
    | --- | --- | --- |
    | NCV | 0.467 | 15.107 |
    | anti-IL23 scFv 1 | 0.440 | 14.055 |
    | anti-IL23 scFv 2 | 0.420 | 13.276 |
    | anti-IL23 scFv 3 | 0.432 | 13.744 |
    | anti-IL23 scFv 4 | 0.499 | 16.354 |
    | anti-IL23 scFv 5 | 0.426 | 13.510 |
  + On **Sep 9**, we proceeded with the western blot. After protein transfer to PVDF membranes, we incubated the membrane overnight at 4 °C with the primary antibody (mouse anti-His tag).
  + On **Sep 10**, we performed several parallel tasks:
    1. **1. Western blot development:** After overnight incubation with primary antibody, the membrane was washed and incubated with HRP-conjugated secondary antibody. A chemiluminescent substrate was added, and the membrane was imaged to observe scFv expression and secretion patterns.
    2. **2. Caco-2 cell maintenance:** We found the Caco-2 cell will grow better after changing the medium. So we changed the medium for the Caco-2 culture. Cell density was examined under the microscope to assess readiness for treatment.
    3. **3. E. coli DH5α culture:** We inoculated E. coli DH5α in selective LB medium to propagate plasmids for future transfection or cloning. Overnight shaking culture was started at 37 °C.
    ![](https://static.igem.wiki/teams/5898/notebook/notebook-figure6.webp)

    **Figure 6. Validation of the scFv expression in HEK293T cells lysate collected by rapidly freezing (in liquid nitrogen) and thawing on Sep 8.**
  + On **Sep 11**, we performed plasmid extraction (miniprep) from overnight E. coli DH5α cultures grown on September 10. Plasmids were purified using a commercial miniprep kit according to standard protocol. After extraction, we measured DNA concentration and purity using Nanodrop spectrophotometry.

    **Table 12. Nanodrop result for plasmid 1 to 5, 3 tubes per plasmids.**

    | Sample | Concentration (ng/µL) | A260/A280 | A260/A230 | A260 | A280 |
    | --- | --- | --- | --- | --- | --- |
    | 1-1 | 347 | 1.87 | 2.22 | 6.94 | 3.72 |
    | 1-2 | 303.6 | 1.86 | 2.26 | 6.07 | 3.26 |
    | 1-3 | 265.9 | 1.86 | 2.18 | 5.32 | 2.87 |
    | 2-1 | 253.6 | 1.86 | 2.20 | 5.07 | 2.73 |
    | 2-2 | 289.7 | 1.86 | 2.20 | 5.79 | 3.12 |
    | 2-3 | 240.3 | 1.86 | 2.24 | 4.81 | 2.58 |
    | 3-1 | 348.5 | 1.86 | 2.23 | 6.97 | 3.74 |
    | 3-2 | 347.6 | 1.87 | 2.26 | 6.95 | 3.73 |
    | 3-3 | 366.3 | 1.86 | 2.24 | 7.33 | 3.93 |
    | 4-1 | 306.3 | 1.86 | 2.17 | 6.13 | 3.30 |
    | 4-2 | 224.2 | 1.85 | 2.14 | 4.48 | 2.43 |
    | 4-3 | 282.2 | 1.86 | 2.20 | 5.64 | 3.04 |
    | 5-1 | 275.1 | 1.86 | 2.20 | 5.50 | 2.97 |
    | 5-2 | 320.8 | 1.86 | 2.24 | 6.42 | 3.45 |
    | 5-3 | 339.8 | 1.86 | 2.26 | 6.80 | 3.65 |
  + On **Sep 12**, we stimulated HEK293T cells with recombinant IL-23 and simultaneously treated them with anti-IL-23 scFv antibodies collected on August 29 for 72 hours. These scFv constructs included multiple prototypes previously verified through Western blot and ELISA. The goal of this experiment was to evaluate the functional activity of the antibodies in neutralizing IL-23–mediated signaling. By mimicking an inflammatory environment with IL-23 stimulation, we aimed to observe downstream changes.
* Sep 15 - Sep 21 (week 38)

  This week focused on functional validation of anti-IL-23 scFv antibodies through IL-23 stimulation, Western blot analysis of downstream signaling (e.g., STAT3, NF-κB, JAK2), and ELISA-based detection.

  + On **Sep 15**, HEK293T cells previously treated with IL-23 and scFv antibodies were harvested for Western blot analysis. Protein lysates were prepared and gels were cast for SDS-PAGE.
  + On **Sep 16**, To investigate the downstream signaling response to IL-23 stimulation, the Western blot membrane from the previous protein extraction was sectioned into three parts and probed for inflammatory pathway targets.

    **Table 13. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB p65, JAK2, p-NF-κB, STAT3, CDK6, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.**

    | Sample | 1°Ab (1:1000) | 2°Ab (1:10000) |
    | --- | --- | --- |
    | 1-1 | p-STAT3 | HRP-Rabbit |
    | 1-2 | NF-κB (p65) | HRP-Rabbit |
    | 1-3 | GAPDH | HRP-Rabbit |
    | 2-1 | p-JAK2 | HRP-Rabbit |
    | 2-2 | p-NF-κB (p-p65) | HRP-Rabbit |
    | 2-3 | CDK6 | HRP-Rabbit |
  + On **Sep 17**, The Western blot was developed and imaged to assess activation of inflammatory signaling pathways in response to IL-23. In parallel, the culture medium was replaced, and internal control antibody staining was performed. On the same day, IL-23 ELISA plates were coated for the next assay.+ On **Sep 18**, When reviewing the setup, we found that PBS buffer had been mistakenly used instead of the proper coating buffer for the ELISA plate. To correct this issue, new IL-23 ELISA plates were re-coated with the correct buffer. Internal controls for Western blot analysis—including JAK2, STAT3, and GAPDH—were also performed. In addition, new Caco-2 cells were subcultured for upcoming experiments, and SDS-PAGE gels were prepared for a Western blot to evaluate the inflammatory response in cells treated with IL-23 for six hours.

      **Table 14. Validation of internal controls (JAK2, STAT3, GAPDH) for Western Blot normalization.**

      | Sample | 1°Ab (1:1000) | 2°Ab (1:10000) |
      | --- | --- | --- |
      | 1-1 | JAK2 | HRP-Rabbit |
      | 2-1 | STAT3 | HRP-Rabbit |
      | 2-3 | GAPDH | HRP-Rabbit |

      ![](https://static.igem.wiki/teams/5898/notebook/notebook-figure7.webp)

      **Figure 7. Functional validation of scFvs in Caco-2 cells co-treated with IL-23 for 72 hours.**
    + On **Sep 19**, we treated Caco-2 cells with 20 μg and 40 μg of IL-23 for 6 hours, respectively, to determine the optimal inflammatory response time. Additionally, we performed ELISA on the supernatant collected on Sep 8 to achieve an ideal OD450 reading. However, the ELISA results did not align with our expectations. The anti-IL-23 scFv #1 showed an inconsistent OD pattern, while no significant signal was detected for the anti-IL-23 scFv p19 or p40. This outcome was unexpected, as previous assays have confirmed that both p19 and p40 possess IL-23 binding capability. Upon review, we identified a technical issue in this ELISA run: the HRP-conjugated secondary antibody for rabbit IgG was mistakenly added instead of the HRP–anti-mouse antibody. The error was promptly corrected by washing the plate and re-adding the proper HRP–anti-mouse antibody. Given this procedural error, the current results are not considered reliable, and the assay will be repeated to validate the findings.

      **Table 15. ELISA quantitative assessment OD450 values of scFv binding activity, collected from culture supernatant on Sep 8.**

      | Supernatant Protein Amount (μg) | 1 | P19 | P40 |
      | --- | --- | --- | --- |
      | 0 | 0.081 | 0.091 | 0.096 |
      | 0.3125 | - | 0.083 | 0.111 |
      | 0.625 | 0.099 | 0.096 | 0.146 |
      | 1.25 | 0.116 | 0.091 | 0.117 |
      | 2.5 | 0.144 | 0.092 | 0.14 |
      | 5 | 0.593 | 0.097 | 0.153 |
      | 10 | 0.091 | - | - |

      ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-8.avif)

      **Figure 8. ELISA quantitative assessment OD450 curves of scFv binding activity, collected on Sep 8.**
    + On **Sep 21**, we coated IL-23 ELISA plates to test the cell lysate collected on Sep 8.
* Sep 22 - Sep 26 (week 39)

  This week, we focused on determining the optimal inflammatory response time of Caco-2 cells and aimed to obtain ideal ELISA OD450 readings.

  + On **Sep 22**, we performed a western blot to assess the inflammatory response of Caco-2 cells treated with IL-23 for 6 hours. Additionally, we measured the OD450 values via ELISA using cell lysate collected on Sep 8. The ELISA performed with the Sep 8 lysate demonstrated a consistent dose-dependent curve, particularly for p19 and p40, suggesting effective dilution and binding. Based on this result, we will re-evaluate the Aug 29 lysate under the same conditions to confirm antibody performance and reproducibility.

    **Table 16. Second ELISA quantitative assessment OD450 values of scFv binding activity, collected from culture supernatant on Sep 8.**

    | Supernatant Protein Amount (μg) | #1 | p19 | p40 | NCV | #2 | #3 |
    | --- | --- | --- | --- | --- | --- | --- |
    | 0 | 0.086 | 0.088 | 0.087 | - | - | - |
    | 1.25 | 0.083 | 0.209 | 0.787 | - | - | - |
    | 2.5 | 0.084 | 0.275 | 0.746 | - | - | - |
    | 5 | 0.085 | 0.341 | 0.819 | - | - | - |
    | 10 | 0.092 | 0.556 | 0.985 | 0.084 | 0.081 | 0.086 |
    | 20 | 0.089 | 0.693 | 1.188 | 0.096 | 0.084 | 0.086 |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-9.avif)

    **Figure 9. Second ELISA quantitative assessment OD450 curves of scFv binding activity, collected from culture supernatant on Sep 8.**
  + On **Sep 23**, we reviewed the Western blot results to assess the inflammatory response of Caco-2 cells treated with IL-23 for 6 hours. Additionally, we subcultured Caco-2 cells for upcoming experiments and coated a new ELISA plate to analyze the lysate collected on Aug 29.

    **Table 17. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB p65, JAK2, p-NF-κB, STAT3, CDK6, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.**

    | Sample | 1°Ab (1:1000) | 2°Ab (1:10000) |
    | --- | --- | --- |
    | 1-1 | p-STAT3 | HRP-Rabbit |
    | 1-2 | NF-κB (p65) | HRP-Rabbit |
    | 1-3 | GAPDH | HRP-Rabbit |
    | 2-1 | p-JAK2 | HRP-Rabbit |
    | 2-2 | p-NF-κB (p-p65) | HRP-Rabbit |
    | 2-3 | GAPDH | HRP-Rabbit |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook-figure10.webp)

    **Figure 10. Validation of the optimal inflammatory response time in Caco-2 cells treated with IL-23 for 6 hours.**
  + On **Sep 24**, we treated Caco-2 cells with IL-23 for 15 minutes, 30 minutes, 1 hour, and 3 hours, respectively, to determine the optimal time point for inflammatory response. Additionally, we performed ELISA and recorded the OD450 values using the cell lysate collected on Aug 29. And we prepared a SDS-PAGE gel for western blot tomorrow.

    **Table 18. OD450 value from ELISA using lysate collected on Aug 29.**

    | Supernatant Protein Amount (μg) | NCV | #1 | #2 | #3 | p19 | p40 |
    | --- | --- | --- | --- | --- | --- | --- |
    | 0 | 0.097 | 0.084 | 0.142 | 0.093 | 0.092 | 0.105 |
    | 6.25 | 0.118 | 0.102 | 0.100 | 0.103 | 0.286 | 0.476 |
    | 12.5 | 0.150 | 0.122 | 0.100 | 0.121 | 0.357 | 0.601 |
    | 25 | 0.127 | 0.122 | 0.110 | 0.146 | 0.483 | 0.882 |
    | 50 | 0.154 | 0.152 | 0.144 | 0.156 | 0.819 | 1.115 |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-11.avif)

    **Figure 11. ELISA quantitative assessment OD450 values of scFv binding activity, collected from cell lysate on Aug 29.**
  + On **Sep 25**, we ran SDS-PAGE for Western blot analysis.

    **Table 19. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB , p-JAK2, p-NF-κB, β actin, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.**

    | Sample | 1°Ab (1:1000) | 2°Ab (1:10000) |
    | --- | --- | --- |
    | 1-1 | p-STAT3 | HRP-Rabbit |
    | 1-2 | NFKB | HRP-Rabbit |
    | 1-3 | GAPDH | HRP-Rabbit |
    | 2-1 | p-JAK2 | HRP-Rabbit |
    | 2-2 | p-NFKB | HRP-Rabbit |
    | 2-3 | β actin | HRP-Rabbit |
  + On Sep 26, we visualized and analyzed the Western blot membranes to assess the expression of inflammatory markers.
    ![](https://static.igem.wiki/teams/5898/notebook/notebook-figure12.webp)

    **Figure 12. Determination of optimal timepoint for IL-23 stimulation in CaCo2 cells.**
* Sep 29 - Oct 5 (week 40)

  In this week, we focused on evaluating the inflammatory response of Caco-2 cells under IL-23 stimulation and assessing the functional effects of anti-IL-23 scFv candidates.

  + On **Sep 29**, Caco-2 cells were subcultured in preparation for IL-23 stimulation and downstream analysis. In addition, ELISA plates were coated with the IL-23 capture antibody in preparation for analyzing the supernatant collected on Aug 29.
  + On **Sep 30**, Cells were switched to serum-free DMEM and stimulated with IL-23 for 30 minutes. Anti-IL-23 scFvs were added for functional testing at the same time. After treatment, cell lysates were collected for Western blot. In parellel, an ELISA was performed using the supernatant we collected on Aug 29. SDS-PAGE gels were prepared for the following day's protein separation.

    **Table 20. OD450 value from ELISA using supernatant collected on Aug 29.**

    | Supernatant Protein Amount (μg) | NCV | #1 | #2 | #3 | #19 | #40 |
    | --- | --- | --- | --- | --- | --- | --- |
    | 0 | 0.092 | 0.075 | - | - | 0.077 | 0.078 |
    | 0.625 | - | - | - | - | - | 0.536 |
    | 1.25 | 0.095 | 0.161 | - | - | 0.238 | 0.492 |
    | 2.5 | 0.085 | 0.11 | - | - | 0.304 | 0.763 |
    | 5 | 0.129 | 0.077 | 0.079 | 0.075 | 0.279 | 0.844 |
    | 10 | 0.09 | 0.088 | 0.083 | 0.077 | 0.366 | 1.1 |
    | 20 | 0.08 | 0.126 | 0.099 | 0.076 | 0.59 | - |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-13.avif)

    **Figure 13. ELISA validation of the binding capability of scFvs collected from cell lysates.**
  + On **Oct 1**, SDS-PAGE was performed to separate proteins from the treated lysates. Proteins were transferred to membranes, which were incubated with primary antibodies targeting key inflammatory markers.

    **Table 21. The Western blot membrane was cut into three sections to probe multiple targets in parallel. Primary antibodies included anti-pSTAT3, NF-κB , p-JAK2, p-NF-κB, and GAPDH. Each blot was incubated with 1° antibodies (1:1000) and appropriate HRP-conjugated 2° antibodies (1:10,000) for detection.**

    | Sample | 1° Antibody (1°Ab) | 2° Antibody (2°Ab) |
    | --- | --- | --- |
    | 1-1 | p-STAT3 | HRP-Rabbit (1:5000) |
    | 1-2 | NFKB | HRP-Rabbit |
    | 1-3 | GAPDH | HRP-Rabbit |
    | 2-1 | p-JAK2 | HRP-Rabbit |
    | 2-2 | p-NFKB | HRP-Rabbit (1:5000) |
    | 2-3 | GAPDH | HRP-Rabbit |
  + On **Oct 2**, Membranes were incubated with HRP-conjugated secondary antibodies and visualized by chemiluminescence. Western blot results were analyzed to assess inflammation-related signaling changes under IL-23 and scFv treatment.
    ![](https://static.igem.wiki/teams/5898/notebook/new-fig-14.avif)

    **Figure 14. . Functional validation of the scFv in CaCo-2 cell treated with IL23 for 30 min.**
  + On **Oct 4**, ELISA was performed to assess the binding performance of anti-IL-23 scFv #1-3 that were produced and purified by a biotechnology company using HEK293 cells.(Note: our lab typically uses HEK293T for scFv expression.) And we used p19 which was collected from culture supernatant on Sep 2 in contrast with these three prototypes. This test was conducted to verify the functional activity of the externally sourced and purified scFvs.

    **Table 22. OD450 value from ELISA using scFv produced by biotechnology company.**

    |  | p19 | #1 | #2 | #3 |
    | --- | --- | --- | --- | --- |
    | 0 | 0.066 | 0.069 | 0.073 | 0.073 |
    | 0.15625 | 0.105 | 0.077 | 0.106 | - |
    | 0.3125 | 0.107 | 0.082 | 0.074 | - |
    | 0.625 | 0.122 | 0.073 | 0.077 | - |
    | 1.25 | 0.159 | 0.078 | 0.072 | - |
    | 2.5 | 0.201 | 0.093 | 0.077 | - |
    | 5 | 0.274 | 0.098 | 0.085 | - |
    | 10 | 0.484 | 0.106 | 0.111 | - |
    | 0.051563 | - | - | - | 0.078 |
    | 0.051563 | - | - | - | 0.078 |
    | 0.103125 | - | - | - | 0.075 |
    | 0.20625 | - | - | - | 0.069 |
    | 0.4125 | - | - | - | 0.071 |
    | 0.825 | - | - | - | 0.075 |
    | 1.65 | - | - | - | 0.078 |
    | 3.3 | - | - | - | 0.088 |

    ![](https://static.igem.wiki/teams/5898/notebook/notebook2-figure-15.avif)

    **Figure 15. ELISA validation of the binding activity of purified scFv1, scFv2, and scFv3 expressed in HEK293 cells.**

{% endblock %}
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{% 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/5898/header/engineering-header.webp)

### Project Aim

This study seeks to establish a probiotic, oral alternative therapy prototype by engineering *Lactobacillus* to secrete a target-specific single-chain variable fragment (scFv) for potential in situ delivery in the gut. As a proof of concept targeting the IL-23, we designed a construct to drive secreted expression in lactobacilli of an anti-IL-23 scFv derived from ustekinumab, and to verify a minimum viable function (“produced, measurable, and specifically antigen-binding”). Prior work supports *Lactobacillus* as a live vector for mucosal delivery and passive immunization using antibody fragments, motivating our application-oriented design.

C1(D)

C1(B)

C1(T)

C1(L)

C2(D)

C2(B)

C2(T)

C2(L)

### Engineering

## Cycle 1: Design (D)

#### Parts / Circuit Design

The target gene is an anti-IL-23 scFv (ustekinumab-derived) arranged as VH–(G4S)₃–VL. To enable secretion, we fused an N-terminal SlpA signal peptide; a C-terminal 6×His tag facilitates detection and downstream purification. The expression backbone is pLEM415 under the ldhL promoter. Practically, we started from the pLEM415-ldhL-mRFP1 plasmid and commissioned removal of the *mRFP1 ORF*, replacing it with a codon-optimized anti-IL-23 scFv while leaving regulatory elements unchanged—thereby minimizing confounders and preserving comparability with existing workflows. Use of pLEM415/ldhL in *Lactobacillus* expression is documented and materials are publicly available.

#### Rationale and Tools

Immunoglobulin variable domains require correct disulfide bond formation to maintain structure and antigen binding; directing the product through the Gram-positive Sec pathway and into the extracellular milieu can favor proper folding and reduce intracellular aggregation. Ustekinumab binds the shared p40 subunit of IL-12/IL-23, blocking receptor engagement; leveraging its variable regions provides biochemical specificity for the IL-23 axis. At the sequence level, we performed codon optimization for *Lactobacillus* and employed standardized cloning overhangs for modular assembly; plasmid maps and junctions were planned with routine molecular design software.

#### Sequence Disclosure (for Reproducibility)

Provide the complete sequences below to enable exact replication. Include both nucleotide (codon-optimized for Lactobacillus) and amino-acid sequences, with clear domain boundaries:

Construct:pLEM415-ldhL-SlpA–anti-IL-23 p40 scFv–6×His

Topology:SlpA SP – VH – (G4S)₃ – VL – 6×His

#### IP / Ethics Notice

The ustekinumab-derived variable regions used in this project are for academic research and educational purposes only. Related sequences/uses may be protected by patents and trademarks held by Janssen Biotech/Johnson & Johnson. Any applications beyond research (e.g., commercialization, clinical use) are outside the scope of this project and require prior authorization from the rights holders.

## Cycle 1: Build (B)

#### Manufacturing

The construct will be built by GentleGen according to our specifications: starting from pLEM415–Idhl–mRFP1, using ApaI (5′) and ClaI (3′) restriction sites to remove the mRFP1 ORF and replace it with a codon-optimized SlpA–VH–(G4S)₃–VL–6×His insert (optimized for *Lactobacillus*). All other regulatory elements (Idhl promoter/RBS/terminator) remain unchanged.

#### Complete annotated map

![](https://static.igem.wiki/teams/5898/engineering/plem415-anti-il-23-scfv.webp)

## Cycle 1・TEST

#### Sequence QC (TEST)

Vendor and in-house verification identified two variants in the scFv CDS: a 1-bp deletion of G at nt 755 and a C→T transition at nt 1163.By aligning Sanger reads ([CX9047\_C10.pdf](https://static.igem.wiki/teams/5898/engineering/cx9047-c10.pdf), [CX9048\_E07.pdf](https://static.igem.wiki/teams/5898/engineering/cx9048-e07.pdf), [M13F-77\_C09.pdf](https://static.igem.wiki/teams/5898/engineering/m13f-77-c09.pdf)) to the codon-optimized reference ([A243050-1full.pdf](https://static.igem.wiki/teams/5898/engineering/a243050-1full.pdf)) using NCBI BLASTn, we confirmed both events. Results revealed a 1-bp G deletion at nt755, producing a frameshift and predicted premature stop, which compromises downstream domains and likely disrupts the C-terminal 6×His tag. We also observed a C→T substitution at nt1163; its protein-level consequence depends on codon context (synonymous vs. missense vs. nonsense) but does not alter the decision once the frameshift is present. Due to the observed deletion mutation (1-bp deletion causing a frameshift), we will not proceed with functional assays for this construct.

#### Sequence QC (Evidence)

To support these findings, we provide BLAST alignment snapshots and Sanger chromatograms:

1. **BLAST**

   ![](https://static.igem.wiki/teams/5898/engineering/engineering1.webp)

   **Figure F1. BLAST alignment at nt755 showing the frameshift-causing deletion.**

   Alignment snapshot around the predefined coordinate (nt755) with Query = Sanger read and Subject = codon-optimized reference CDS. The dash (–) indicates a single-base gap relative to the reference, consistent with a 1-bp deletion in the insert that would shift the reading frame and predict a downstream premature stop.

   ![](https://static.igem.wiki/teams/5898/engineering/engineering2.webp)

   **Figure F2. BLAST alignment at nt1163 showing the C→T single-nucleotide substitution.**

   Alignment snapshot around the predefined coordinate (nt1163) with Query = Sanger read and Subject = codon-optimized reference CDS. A single base differs between the two sequences (C→T in the read), consistent with the reported SNP at nt1163.
2. **Sanger**

   ![](https://static.igem.wiki/teams/5898/engineering/engineering4.webp)

   **Figure F3. Sanger chromatogram showing the single-base deletion signature (nt755 region).**

   ![](https://static.igem.wiki/teams/5898/engineering/engineering3.webp)

   **Figure F4. Sanger chromatogram confirming the C→T single-nucleotide substitution (nt1163 region).**

## Cycle 1・LEARN

#### Observations

During *E. coli* amplification, the insert repeatedly accumulated InDels (including a 1-bp deletion at nt755) and point mutations, preventing recovery of an intact construct. The issue persisted even with low-recombination strains, indicating sequence intrinsic and context-dependent instability.

#### Hypothesized Causes

Sequence-level: the scFv cassette likely contains homopolymers and/or micro-repeats, promoting polymerase slippage and recombination.

Context effects: within the pLEM415–IdhL framework, local transcriptional read-through, long recombination-prone stretches, or expression burden may increase instability.

#### Decision

To avoid further delays, we will pivot to a mammalian system for functional validation while re-engineering the bacterial construct for stability, then back-port it to *Lactobacillus* once hardened.

#### Expansion into Cycle 2 (broader candidate pool)

To reduce single-sequence risk and enrich the study, we added three de novo scFv candidates (1/2/3) plus a risankizumab-derived anti-IL-23 p19 scFv and an ustekinumab-derived anti-IL-23 p40 scFv, all expressed from pTwist-CMV (mammalian) for the second round. This covers p19 (risankizumab-derived) and p40 (ustekinumab-derived) epitopes, improving our odds and widening optimization space.

#### Reference

1. del Rio, B., Redruello, B., Fernandez, M., Martin, M. C., Ladero, V., & Alvarez, M. A. (2019). Lactic Acid Bacteria as a Live Delivery System for the *in situ* Production of Nanobodies in the Human Gastrointestinal Tract. *Frontiers in Microbiology*, 9, 3179.
2. Bao, S., Zhu, L., Zhuang, Q., Wang, L., Xu, P. X., Itoh, K., Holzman, I. R., & Lin, J. (2013). Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats. *PloS* one, 8(3), e60007.
3. Klotz, C., & Barrangou, R. (2018). Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications. Frontiers in microbiology, 9, 2264.

## Cycle 2・Design (D)

#### Parts / Circuit Design

The targets for Cycle 2 are five secreted scFv candidates expressed in HEK293T: three de novo scFv (1/2/3), a risankizumab-derived anti-IL-23 p19 scFv and an anti-IL-23 p40 scFv. Each construct follows the same topology Igκ leader–VH–(G4S)₃–VL–6×His, driven by the CMV promoter on a pTwist-CMV backbone with a standard polyA signal. Human codon optimization is applied; the C-terminal 6×His tag supports detection (anti-His Western) and optional Ni-NTA cleanup.

#### Rationale and Tools

Secretion via the Igκ signal peptide routes the scFv through the ER/Golgi for oxidative folding and disulfide formation, improving the chance of functional paratopes relative to bacterial cytosol. HEK293T enables rapid, high-level transient expression to generate clean supernatants for binding/neutralization assays. Design steps include human-preferred codons and standardized junctions to keep modules swappable. Plasmid maps and junctions are planned with routine tools, keeping all candidates in a single, comparable expression context (CMV/Igκ/(G4S)₃/His).

#### Sequence Disclosure (for Reproducibility)

Provide full nucleotide (human codon optimized) and amino-acid sequences with labeled domain boundaries.

* Construct(s):
  1. pTwist-CMV–Igκ–scFv 1–6×His
  2. pTwist-CMV–Igκ–scFv 2–6×His
  3. pTwist-CMV–Igκ–scFv 3–6×His
  4. pTwist-CMV–Igκ–anti-IL-23 p19 scFv(risankizumab-derived)–6×His
  5. pTwist-CMV–Igκ–anti-IL-23 p40 scFv(ustekinumab-derived)–6×His
* Topology: Igκ SP – VH – (G4S)₃ – VL – 6×His – Stop codon

#### IP / Ethics Notice

Any ustekinumab-derived and risankizumab-derived variable regions are used for academic research and education only. Related sequences/uses may be protected by the rights holders. Applications beyond research (e.g., commercialization, clinical use) are out of scope and require prior authorization.

## Cycle 2: Build (B)

#### Manufacturing

The scFv CDS (human codon optimized) will be synthesized and assembled by Twist Bioscience directly into the pTwist-CMV backbone downstream of the Igκ signal peptide, following the shared topology Igκ–VH–(G4S)₃–VL–6×His. All regulatory elements (CMV promoter/leader, polyA/terminator, selection marker) remain unchanged to minimize confounders and enable fair comparison across candidates.

#### Complete annotated map

1. pTwist-CMV–Igκ–scFv 1–6×His

   ![](https://static.igem.wiki/teams/5898/engineering/anti-il-23-scfv-1.webp)
2. pTwist-CMV–Igκ–scFv 2–6×His

   ![](https://static.igem.wiki/teams/5898/engineering/anti-il-23-scfv-2.webp)
3. pTwist-CMV–Igκ–scFv 3–6×His

   ![](https://static.igem.wiki/teams/5898/engineering/anti-il-23-scfv-3.webp)
4. pTwist-CMV–Igκ–anti-IL-23 p19 scFv(risankizumab-derived)–6×His

   ![](https://static.igem.wiki/teams/5898/engineering/anti-il-23-scfv-risankizumab-derived.webp)
5. pTwist-CMV–Igκ–anti-IL-23 p40 scFv(ustekinumab-derived)–6×His

   ![](https://static.igem.wiki/teams/5898/engineering/anti-il-23-scfv-ustekinumab-derived.webp)

## Cycle 2: Test (T)

#### Objectives

1. Determine whether each scFv is secreted by HEK293T (CMV–Igκ–VH–(G4S)₃–VL–6×His).
2. Assess binding to IL-23 by ELISA.

#### Methods (concise)

* **Expression:** pTwist-CMV constructs; transient HEK293T (48–72 h).
* **Samples:** collect supernatant and cell lysate separately.
* **Western blot:** reducing SDS-PAGE; anti-His detection; equal loading and identical exposure across lanes.
* **ELISA:** IL-23 coated plates; serial dilutions of supernatant/lysate; anti-His-HRP detection; background-subtracted.

#### Results

1. **Secretion & Binding Overview**

   anti-IL-23 p19 scFv and anti-IL-23 p40 scFv show robust secretion and binding, while anti-IL-23 scFv-1/2/3 are expressed intracellularly (bands at ~25 kDa in lysate) but not secreted. Supernatant Western blots display clear bands at ~50 kDa for p19/p40 and none for #1/2/3; lysate Westerns show ~50 kDa bands for p19/p40 and ~25 kDa bands for #1/2/3, indicating expression without secretion for the scFv-1/2/3. Consistently, supernatant ELISA curves are dose-dependent for p19/p40 (p40 strongest), whereas scFv 1 remains in the background. Lysate ELISA confirms p19/p40 retain IL-23 binding activity in cell extracts; scFv 1/2/3 stay near baseline.
2. **Figure**

   ![](https://static.igem.wiki/teams/5898/engineering/engineering5.webp)

   **Figure C2-WB-SN. Anti-His Western of HEK293T supernatants.**

   Strong bands at ~50 kDa for p19 and p40 indicate efficient secretion; scFv-1/2/3 show no detectable supernatant bands. Reducing conditions; equal loading; identical exposure.

   ![](https://static.igem.wiki/teams/5898/engineering/engineering6.webp)

   **Figure C2-WB-Lysate. Anti-His Western of HEK293T lysates.**

   p19/p40 show bands at ~50 kDa; scFv-1/2/3 show intracellular bands at ~25 kDa, confirming expression but secretion bottlenecks.

   ![](https://static.igem.wiki/teams/5898/engineering/engineering-figure-c2-1.avif)

   **Figure C2-ELISA-SN. Binding of secreted scFv to IL-23 (supernatant).**

   Dose–response curves: p40 strongest (OD450 ≈ 1.2 at 20 µg), p19 positive (≈ 0.7), scFv-1 at background. Same plate/conditions; background-subtracted.

   ![](https://static.igem.wiki/teams/5898/engineering/engineering-figure-c2.avif)

   **Figure C2-ELISA-Lysate. Binding of cell lysates to IL-23 (lysate).**

   Dose–response curves: p40 (≈ 1.15 at 50 µg) and p19 (≈ 0.8 at 50 µg) remain positive; scFv-1/2/3 near background; NCV baseline. Background-subtracted; same detection.

#### What we will do next

p19-scFv & p40-scFv — proceed to functional characterization

Pathway inhibition: Run IL-23–induced pSTAT3 assay (reporter or phospho-flow) on Caco-2 cell model.

scFv-1 / scFv-2 / scFv-3 — collaborate with biomedical company

External production confirmation by Leadgene Biomedical:After reaching out, Leadgene Biomedical has successfully expressed scFv-1/2/3 in HEK293. We will document their expression proof (report/traces) on the LEARN part. This external success indicates that the sequences are producible in HEK cells and supports the hypothesis that our in-house non-secretion stems from chassis rather than an absolute misfolding defect.

![](https://static.igem.wiki/teams/5898/engineering/engineering9.webp)

**Figure C2-anti-IL-23 scFv 1/2/3 expression result (Leadgen biomedical).**

## Cycle 2: Learn (L)

#### What we observed (quantitative summary)

* Secretion vs. expression. In HEK293T (adherent), p19/p40 produced clear supernatant bands (~50 kDa) and dose-dependent IL-23 binding. scFv-1/2/3 showed lysate bands only (~25 kDa) and no supernatant signal, with ELISA near the background.
* External replication. Leadgene reported successful HEK293 (suspension) expression of scFv-1/2/3 under their platform, indicating that these sequences are expressible/secreted in a different 293 context.

#### Why HEK293T (adherent) failed while HEK293 (suspension) worked — model and literature

1. Host format & medium: Suspension-adapted HEK293 variants (e.g., HEK293F/293-3F6) in serum-free, chemically defined media routinely achieve higher titers of secreted proteins and antibodies in transient runs, owing to higher viable cell densities and media/additive optimization for secretion and oxidative folding. Multiple studies document robust, scalable transient production of secreted proteins/antibodies in suspension HEK293 systems.
2. Lineage differences: HEK293T carries SV40 large T antigen and is widely used for transient expression, but suspension 293 derivatives are frequently optimized for secretory outputs; depending on construct context (leader/order/linker/tag), secretion can be superior in suspension lines despite strong intracellular expression in 293T. Comparative and review articles note distinct behaviors among HEK293 sublines (293, 293T, 293F, 293E), including secretion performance and bioprocess suitability.
3. Adherent↔suspension rewiring: Global transcriptomic/proteomic analyses show systematic differences between adherent and suspension HEK293, including lipid/cholesterol metabolism and secretory pathway components—factors that can modulate ER-to-Golgi trafficking, glycosylation, and secretion efficiency. This provides a mechanistic basis for “same CDS, different secretion outcome” across hosts.
4. Antibody formats in 293 systems: Transient antibody/scFv formats have been repeatedly optimized in HEK293 backgrounds (including scFv-Fc and secreted domains), supporting the general claim that 293 suspension platforms are suitable antibody hosts.

#### Reference

1. Jäger, V., Büssow, K., Wagner, A., Weber, S., Hust, M., Frenzel, A., & Schirrmann, T. (2013). High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC biotechnology, 13, 52.
2. Malm, M., Saghaleyni, R., Lundqvist, M. et al. Evolution from adherent to suspension: systems biology of HEK293 cell line development. Sci Rep 10, 18996 (2020).
3. Lao, T., Farnos, O., Bueno, A., Alvarez, A., Rodríguez, E., Palacios, J., Luz, K. R. d. l., Kamen, A., Carpio, Y., & Estrada, M. P. (2023). Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study. Biomedicines, 11(11), 3050.

{% endblock %}
{% block scripts %}
{% 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/5898/header/experiment-header.webp)

☰

#### Experiments

* [Bacterial Work](#section1)
* [Mammalian Cell Work](#section2)
* [Protein Experiments](#section3)

#### Experiments

* [Bacterial Work](#section1)
* [Mammalian Cell Work](#section2)
* [Protein Experiments](#section3)

## Bacterial Work

## Agar Plates

*In this protocol, you can find how to prepare LB agar plates supplemented with ampicillin for bacterial growth and selection.*

**Materials:**

* LB Broth (25g/L)
* Agar (1.5%)
* Ampicillin (100 mg/mL stock solution; 150μg/mL working concentration)

**Protocols:**

1. Prepare 150 mL LB Broth with 1.5% agar.
2. Cool the medium to ~45 °C.
3. Add 225 µL Ampicillin (100 mg/mL stock) to reach a working concentration of 150 µg/mL.
4. Pour plates and store at 4 °C.

## Colony Picking and Starter Culture

*In this protocol, you can find how to pick single colonies and expand them into liquid LB medium for plasmid propagation.*

**Materials:**

* LB agar plates with single colonies (Ampicillin 150 µg/mL)
* LB broth (25g/L)
* Ampicillin (100 mg/mL stock solution; 150 µg/mL working concentration)
* 1.5 mL microcentrifuge tubes (Eppendorf)
* 4 mL culture tubes / vials
* Shaking incubator (37 °C, ~200 rpm)

**Protocols:**

1. Pick a single colony from a selective LB agar plate.
2. Inoculate into 50µL LB broth with ampicillin (150 µg/mL) in a microcentrifuge tube.
3. Incubate at 37 °C with shaking for 3–6 h.
4. Transfer the starter culture into 4 mL LB broth with ampicillin (150 µg/mL).
5. Continue shaking incubation until sufficient growth is reached.

## Plasmid Extraction (pLEM415 / pTwist CMV)

*In this protocol, you can find how to isolate plasmid DNA from transformed E. coli DH5α cultures using a silica column-based miniprep kit.*

**Materials:**

* Overnight culture of transformed E. coli (grown in LB + antibiotic)
* Plasmid Miniprep Kit (silica column based)
* Centrifuge (≥12,000 × g)
* Microcentrifuge tubes (1.5 mL)
* Nuclease-free water or TE buffer (for elution)

**Protocols:**

1. Centrifuge the culture tube at 6000 rpm, 4°C for 15 min, then discard the supernatant.
2. Reaction components (depending on culture volume):

   | LB media | RB | LB | NB | Our experiment |
   | --- | --- | --- | --- | --- |
   | ≤5mL | 250µL | 250µL | 350µL | ✓ |
   | 5-10mL | 500µL | 500µL | 700µL |  |
   | 10-15mL | 750µL | 750µL | 1050µL |  |
   | 15-20mL | 1000µL | 1000µL | 1400µL |  |
3. Add appropriate volume of RB (premixed with RNase A) to the cell pellet and resuspend it completely by pipetting.
4. Add appropriate volume of LB (Blue), immediately mix by inverting the tube 4-6 times (the color of the lysate should change from opaque to bright blue).
5. Add appropriate volume of NB (Yellow), mix by inverting the tube 5-6 times. (The color of the lysate will turn into yellow when the neutralization is complete and a yellowish precipitate will form). Incubate the lysate at room temperature for 2 minutes.
6. Centrifuge at 12,000 × g for 5 minutes, gently transfer the supernatant to a spin column. Centrifuge at 12,000 × g for 1 minute and discard the flow-through (for supernatant more than 800µL, repeat the process once).
7. Add 250µL TB, incubate at room temperature for 10 minutes. Centrifuge at 12,000 × g for 1 minute and discard the flow-through.
8. Add 650µL of WB (check to make sure that ethanol has been added) to the column, Centrifuge at 12,000 × g for 1 minute. Discard the flow through.
9. Centrifuge the column at 12,000 × g for 1-2 minutes to remove residual WB completely.
10. Place the spin column in a clean microcentrifuge tube, add 30-50µL of Elution Buffer or sterile, distilled water directly to the center of the column matrix for higher yield, prewarm EB or ddH₂O at 60-70°C.
11. 10. Centrifuge the column at 10,000 × g for 1 minute to elute DNA. Isolated plasmid DNA is ready to use or can be stored at -20°C.

**\*Notes:**

The following protocol was referenced from the AllPure Plasmid MiniPrep Kit manual provided by the manufacturer.

## Transformation of *E. coli* DH5α Cells (by Heat Shock)

*This protocol describes how to transform competent E. coli cells by introducing plasmid DNA, followed by selection on antibiotic-containing agar plates.*

**Materials:**

* Sterile water
* Plasmid DNA (stock solution, or dried DNA resuspended in Tris-EDTA buffer; e.g., 2 µg dried DNA in 20 µL TE)
* Competent *E. coli* DH5α cells (25 µL per reaction)
* LB broth (without antibiotics)
* LB agar plates with 150 µg/mL ampicillin
* 42 °C water bath
* Ice bucket
* Sterile spreader or triangular glass rod
* Bunsen burner (for sterilization)

**Protocols:**

1. Dilute plasmid DNA to 100 ng/µL.
2. Add plasmid to thawed competent *E. coli* DH5α cells and incubate on ice for 20–30 min.
3. Heat shock at 42 °C for 1 min, then place on ice for 2 min.
4. Add 500 µL LB and recover at 37 °C, 180 rpm for 50 min.
5. Plate 50 µL on LB-Amp agar plate.
6. Incubate overnight at 37 °C to obtain colonies.

## Mammalian Cell Work

## Growth Media

*This section describes the different media used in our project for bacterial and mammalian cell culture.*

1. **DMEM basal**

   *In this protocol, you can find how to prepare and use Dulbecco’s Modified Eagle Medium (DMEM basal) for Caco-2 culture and stimulation experiments.*

   * Composition: DMEM (with 4.5 g/L glucose, L-glutamine and phenol red indicator; without sodium pyruvate)
   * Applications: Caco-2 maintenance, IL-23 stimulation/neutralization assays
2. **DF-12 complete**

   *In this protocol, you can find how to prepare complete DF-12 medium.*

   * Composition: DF-12 (DMEM/F-12) basal medium
   * Supplementation: 10% FBS, Sodium Pyruvate, L-Glutamine and PSA (Penicillin–Streptomycin–Amphotericin B)
   * Applications: HEK293T culture
3. **Opti-MEM**

   *In this protocol, you can find how we use Opti-MEM reduced-serum medium during transfection experiments.*

   * Composition: Opti-MEM Reduced Serum Medium
   * Applications: transient transfection, medium exchange after X2–DNA complex formation

**\*Notes:**

Cells were routinely maintained in complete DF-12 medium. Prior to protein analysis (e.g., SDS-PAGE/Western blot), cultures were washed twice with PBS to remove residual serum proteins.

## Cell Subculturing

*In this protocol, you can find how to subculture adherent mammalian cells (e.g., HEK293T, Caco-2) while maintaining viability for downstream applications.*

**Materials:**

* Complete growth medium ( HEK293T: DF-12 + 10% FBS, [see Growth Media page](#Growth-Media); Caco-2: DMEM)
* PBS (Ca²⁺/Mg²⁺-free)
* Trypsin-EDTA solution
* 15 mL sterile conical tubes
* cell counter
* Trypan Blue solution (0.4%)
* Pipettes, serological pipettes, centrifuge

**Protocols:**

1. Aspirate culture medium from the cells.
2. Wash cells once with 2 mL PBS, then aspirate completely.
3. Add 350–400 µL trypsin-EDTA to cover the cell layer.
4. Incubate at 37 °C for 1–2 min until cells round up and detach.
5. Confirm under microscope that cells are separated into single cells (no sharp edges); gently tap flask if needed.
6. Add 800–1000 µL complete medium (DF-12 + 2% FBS) to neutralize trypsin.
7. Transfer the suspension to a 15 mL conical tube.
8. Centrifuge at 300 × g for 3–5 min.
9. Aspirate and discard supernatant.
10. Flick the pellet and resuspend in fresh complete medium.
11. Mix 30 µL Trypan Blue with 10 µL cell suspension and load onto a hemocytometer.
12. Count viable cells and calculate seeding density.
13. Seed cells into new culture vessels with appropriate volume of fresh medium.
14. Return to the incubator (37 °C, 5% CO₂) and culture under standard conditions.

## Transfection of HEK293T Cells (using NTR II / X2 reagent)

*In this protocol, you can find how to transfect plasmid DNA into HEK293T cells using NTR II / X2 transfection reagent.*

**Materials:**

* HEK293T cells (about 80% confluency)
* Plasmid DNA (endotoxin-free)
* NTR II / X2 transfection reagent

  | transfection reagent | NTR II | X2 |
  | --- | --- | --- |
  | Growth medium | DF-12 | opti-MEM |
  | Complete growth medium | DF-12 + 2% FBS | opti-MEM |
* Sterile microcentrifuge tubes
* Shaking centrifuge (brief spins)

**Protocols:**

1. Prepare plasmid DNA solution: dilute 5µg plasmid (~10µL) in 180 µL growth medium (DF-12 or opti-MEM).
2. Add 10µL NTR II reagent to each DNA/DF-12 + 2% FBS mixture or add 10µL X2 reagent to each DNA/opti-MEM mixture.
3. Gently mix by pipetting or inversion, briefly spin down.
4. Incubate the mixture at room temperature for 25–30 min to allow DNA–NTR II or DNA–X2 complexes to form.
5. Add the DNA–NTR II or DNA–X2 complexes dropwise onto HEK293T cells cultured in 1.8 mL complete growth medium (DF-12 + 2% FBS or opti-MEM) in the dish.
6. Return cells to the incubator (37 °C, 5% CO₂) and culture under standard conditions.

**\*Notes:**

**Serum condition:** Although the manufacturer recommends forming complexes in serum-free medium, in our hands **NTR II–DNA complexes prepared and used directly in DF-12 + 2% FBS** yielded successful transfection in HEK293T. If efficiency drops or cytotoxicity increases,  **switch to serum-free medium for complex formation** and/or reduce serum during the first 4–6 h, then return to DF-12 + 2% FBS.

## Cell Treatments (IL-23 Stimulation)

*In this protocol, you can find how to stimulate Caco-2 cells with IL-23 to identify activation conditions by monitoring downstream signaling.*

**Materials:**

* Caco-2 cells (70–90% confluence)
* DMEM basal medium (without serum)
* Recombinant IL-23 (20 ng/mL; 40 ng per dish)
* PBS (ice-cold)
* CO₂ incubator (37 °C, 5% CO₂)

**Protocols:**

1. Aspirate culture medium from Caco-2 cells.
2. Wash cells 2 times with 2–3 mL cold PBS.
3. Add 2 mL serum-free DMEM basal to each dish.
4. Add IL-23 (20 ng/mL; 40 ng per dish) directly to cells.
5. Incubate cells at 37 °C, 5% CO₂ for different stimulation times: 15, 30, 60, 180 min.
6. At the designated time point, immediately harvest cells:
   * Place dish on ice.
   * Aspirate medium.
   * Wash twice with cold PBS.
   * Add lysis buffer (NETN + inhibitors).
   * Scrape or pipette to collect cells into cold microtubes.
7. Store lysates on ice or proceed directly to Western blot analysis.

## IL-23 Neutralization by Anti-IL-23 scFv

*In this protocol, you can find how to test whether His-tag scFvs block IL-23-induced signaling in Caco-2 cells.*

**Materials:**

* Caco-2 cells (grown to ~80% confluence)
* DMEM basal medium (serum-free)
* PBS (ice-cold)
* Anti-IL-23 scFv (40 µg/mL; 80 µg per dish; NCV, #1, p19, p40)
* Recombinant IL-23 (20 ng/mL; 40 ng per dish)
* CO₂ incubator (37 °C, 5% CO₂)

**Protocols:**

1. Aspirate culture medium from Caco-2 cells.
2. Wash cells 2 times with 2–3 mL cold PBS.
3. Add 2 mL serum-free DMEM basal to each dish.
4. In the biosafety cabinet, add scFv (40 µg/mL; 80 µg per dish) directly to cells (test NCV, #1, p19, p40).
5. Add IL-23 (20 ng/mL; 40 ng per dish) into the same dish after scFv addition.
6. Incubate cells at 37 °C, 5% CO₂ for 30 min.
7. Harvest cells immediately for downstream lysis and Western blot analysis.

## Protein Experiments

## Cell Lysis (NETN)

*In this protocol, you can find how to lyse cells prior to SDS–PAGE/Western blot.*

**Materials:**

* NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris‑HCl pH 8.0, 0.5% NP‑40)
* Protease & phosphatase inhibitor cocktails
* Ice, microtubes, centrifuge

**Protocols:**

1. Wash cells with cold PBS; add NETN and inhibitors on ice.
2. Vortex and incubate 5 min on ice, repeat 3 times.
3. Spin 12,000 rpm, 15 min, 4 °C; collect supernatant as lysate for quantification.

## Cell Lysis (Freeze and thaw)

*In this protocol, you can find how to lyse cells prior to SDS–PAGE/Western blot using freeze–thaw cycles.*

**Materials:**

* PBS (ice-cold)
* Protease & phosphatase inhibitor cocktails
* Dry ice/ethanol bath or −80 °C freezer
* 25 °C dry bath
* Microtubes, centrifuge

**Protocols:**

1. Wash cells with cold PBS and collect into a microtube. Add protease/phosphatase inhibitors.
2. Immerse the tube in liquid nitrogen for 10–20 sec to freeze cells completely.
3. Thaw the tube in a 25 °C dry bath for 30–60 sec until fully liquid.
4. Repeat steps 2–3 for a total of 10 cycles.
5. Centrifuge at 12,000 rpm, 15 min, 4 °C to pellet debris.
6. Collect the supernatant as lysate for quantification or downstream analysis.

## Supernatant Concentration

*In this protocol, you can find how to concentrate culture supernatant (sup) using centrifugal filter units for downstream protein analysis.*

**Materials:**

* Collected culture supernatant (after PBS wash and Opti-MEM incubation)
* Centrifuge tubes (15 mL, sterile)
* 0.45 µm sterile syringe filter
* Amicon Ultra-0.5 centrifugal filter units, 3 kDa MWCO
* Tabletop centrifuge (fixed-angle rotor, capable of ≥14,000 × g)
* Ice

**Protocols:**

1. Collect culture supernatant and centrifuge at 1500 × g for 5 min to remove cell debris.
2. Filter the clarified supernatant through a 0.45 µm filter to remove remaining cells and large particulates.
3. Transfer clarified supernatant into an Amicon Ultra-0.5 (3 kDa) centrifugal filter unit.
4. Centrifuge at 14,000 × g, 4 °C until the desired final volume is reached (e.g., concentrate from 500 µL to 100 µL), usually 15-20 mins.
5. Recover concentrated supernatant from the filter unit by inverting the column into a clean tube and centrifuging at 1,000 × g for 2 min.
6. Keep concentrated samples on ice for immediate use or store at –80 °C.

## Protein Quantification (BCA, microplate)

*In this protocol, you can find how to quantify protein concentration using a BCA assay at 562 nm.*

**Materials:**

* BCA Protein Assay Kit (working reagent)
* BSA standards (serial dilution)
* 96‑well clear plate; plate reader (λ = 562 nm)

**Protocols:**

1. Prepare BSA standards and dilute lysates in the same buffer.
2. Add X µL sample/standard + Y µL BCA working reagent per well (follow kit).
3. Incubate 37 °C, 30 min; read A562.
4. Generate standard curve; calculate sample concentrations.

## Sample Preparation for SDS–PAGE

*In this protocol, you can find how to prepare quantified lysates for electrophoresis.*

**Materials:**

* 5 × Laemmli sample buffer (Tris-HCl pH6.8, SDS, β-ME, glycerol, Bromophenol Blue)
* NETN or PBS

**Protocols:**

1. Mix protein lysate with 5× sample buffer to a final 1× concentration, and use NETN/PBS to adjust volumes so that all samples have the same final volume.
2. Heat at 95 °C for 5 min to denature proteins.
3. Cool on ice and briefly spin down.
4. Load ~20 µg protein per lane for SDS–PAGE
5. If the gel will be run the next day, store prepared samples at −20 °C until use.

## SDS PAGE

*In this protocol, you can find how to separate proteins by size using SDS–PAGE.*

**Materials:**

* Protein samples
* hand-cast SDS–PAGE gels (12% resolving gel with 5% stacking gel)
* Protein ladder (molecular weight marker)
* Running buffer (1× Tris-Glycine-SDS)
* Electrophoresis apparatus and power supply

**Protocols:**

1. Assemble SDS–PAGE gel in electrophoresis tank and add 1× running buffer.
2. Load protein samples and markers into wells.
3. Run at 85 V through stacking gel, then increase to 130 V until the dye front reaches the bottom.
4. Stop electrophoresis and prepare for transfer.

## Western Blot

*In this protocol, you can find how to transfer proteins to a membrane and detect them by antibody-based chemiluminescence.*

**Materials:**

* PVDF (requires methanol activation)
* Transfer buffer
* Filter papers and sponges
* Blocking buffer (5% milk in TBST)
* Primary antibody (e.g., p-STAT3 Y705, His-tag…)
* Loading control antibody (e.g.,GAPDH)
* HRP-conjugated secondary antibody
* TBST (TBS + 0.1% Tween-20)
* ECL substrate
* Imaging system (chemiluminescence detection)

**Protocols:**

1. Assemble transfer sandwich (sponge–filter paper–gel–membrane–filter paper–sponge).
2. Transfer proteins to the membrane (e.g., wet transfer at 100 V for 150 min , 4 °C).
3. Block membrane with 5% milk in TBST for 1h at room temperature.
4. Incubate with primary antibody (overnight at 4 °C).
5. Wash membrane 3 × 6 min with TBST.
6. Incubate with HRP-conjugated secondary antibody (60 min at RT).
7. Wash membrane 3 × 6 min with TBST.
8. Add ECL substrate and detect bands with imaging system.
9. If additional antibodies need to be tested, the membrane can be stripped for 30 minutes before reprobing.

## ELISA

*In this protocol, you can find how to detect His-tagged anti-IL-23 scFv by ELISA.*

**Materials:**

* High-binding 96-well ELISA plate
* ELISA coating buffer (100mM NaHCO₃ pH 9.6)
* Recombinant IL-23 (5 ng/well in 50 µL coating buffer)
* PBS-T (PBS + 0.1% Tween-20)
* Blocking buffer: 5% milk in PBS
* His-tagged scFv samples (serial dilutions in PBS-T)
* Mouse anti-His antibody (1°; ~1:500 in 5% milk)
* Anti-mouse-HRP antibody (2°; 1:5000 in 5% milk)
* TMB substrate (100 µL/well)
* 1 N HCl (50 µL/well)
* Plate reader (OD 450 nm)

**Protocols:**

1. Coat wells with IL-23 (5 ng/well in 50 µL coating buffer) and incubate overnight at 4 °C.
2. Remove coating solution and wash wells 3× with 200µL PBS-T per well (1 min each).
3. Add 100 µL/well blocking buffer (5% milk in PBS), incubate 37 °C for 1 h.
4. Remove blocking solution, and wash wells 3× with 200µL PBS-T per well .
5. Add 50 µL/well scFv samples (serial 2-fold dilutions, e.g. 5 → 2.5 → 1.25 → 0.625 → 0.3125 µg/mL), incubate 37 °C for 1 h.
6. Wash wells 3× with 200µL PBS-T per well.
7. Add 50 µL/well mouse anti-His antibody (diluted ~1:500 in 5% milk), incubate 37 °C for 1 h.
8. Wash wells 3× with 200µL PBS-T per well.
9. Add 100 µL/well anti-mouse-HRP antibody (1:5000 in 5% milk), incubate 37 °C for 1 h.
10. Wash wells 3× with 200µL PBS-T per well.
11. Add 100 µL/well TMB substrate, incubate RT for 5–15 min until color develops.
12. Add 50 µL/well 1 N HCl to stop the reaction.
13. Measure absorbance at 450 nm.

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{% 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/5898/header/results-header.webp)

## Evaluation of scFv Expression Feasibility in *Lactobacillus*

Vendor and in-house verification identified two variants in the scFv CDS: a 1-bp deletion of G at nt 755 and a C→T transition at nt 1163.By aligning Sanger reads to the codon-optimized reference using NCBI BLASTn, we confirmed both events. Results revealed a 1-bp G deletion at nt755, producing a frameshift and predicted premature stop, which compromises downstream domains and likely disrupts the C-terminal 6×His tag. We also observed a C→T substitution at nt1163; its protein-level consequence depends on codon context (synonymous vs. missense vs. nonsense) but does not alter the decision once the frameshift is present. Due to the observed deletion and mutation (1-bp deletion causing a frameshift), we will not proceed with functional assays for this construct.

### **Sequence QC (Evidence)**

To support these findings, we provide BLAST alignment snapshots and Sanger chromatograms:

1. BLAST

   ![](https://static.igem.wiki/teams/5898/engineering/engineering1.webp)

   **Figure 1. BLAST alignment at nt755 showing the frameshift-causing deletion.**

   Alignment snapshot around the predefined coordinate (nt755) with Query = Sanger read and Subject = codon-optimized reference CDS. The dash (–) indicates a single-base gap relative to the reference, consistent with a 1-bp deletion in the insert that would shift the reading frame and predict a downstream premature stop.

   ![](https://static.igem.wiki/teams/5898/engineering/engineering2.webp)

   **Figure 2. BLAST alignment at nt1163 showing the C→T single-nucleotide substitution.**

   Alignment snapshot around the predefined coordinate (nt1163) with Query = Sanger read and Subject = codon-optimized reference CDS. A single base differs between the two sequences (C→T in the read), consistent with the reported SNP at nt1163.
2. Sanger

   ![](https://static.igem.wiki/teams/5898/results/engineering-figure-f3.avif)

   **Figure 3. Sanger chromatogram showing the single-base deletion signature (nt755 region).**

   ![](https://static.igem.wiki/teams/5898/results/engineering-figure-f4.avif)

   **Figure 4. Sanger chromatogram confirming the C→T single-nucleotide substitution (nt1163 region).**

## Verification of scFv Expression (in HEK293T)

To confirm that our constructs were successfully expressed in mammalian cells, we transfected HEK293T cells with five different anti-IL-23 scFv prototypes. Among them, three were originally designed antibodies, generated by assembling sequences identified through extensive database research, while the other two were scFv versions of clinically used antibody drugs. After 48h, cell lysates were analyzed by Western blot using an anti-His antibody. We observed clear bands at ~25 and ~50 kDa, corresponding to the expected molecular weight of the scFv. These results confirmed that our constructs were expressed correctly in HEK293T cells.

![](https://static.igem.wiki/teams/5898/results/result-figure-5.avif)

**Figure 5. Validation of scFv expression in cell lysates.**

## Assessment of scFv Secretion

To evaluate whether our constructs were secreted into the extracellular medium, we analyzed both the supernatants from our own HEK293T transfection and the purified proteins expressed by an outsourced HEK293 expression system.

In our lab-scale HEK293T transfection experiments, supernatants were collected after transfection and concentrated using Amicon Ultra-0.5 centrifugal filters (3 kDa cutoff) to enhance detection. Western blot analysis revealed that **only the two scFv constructs derived from clinically used antibody drugs were detected in the supernatant**, while the three originally designed prototypes showed no visible secretion signals. Interestingly, Western blot analysis of the cell lysates revealed both full-length and cleaved forms of the prototype scFv, suggesting that **the signal peptide might have been prematurely removed**, preventing proper secretion.

However, when the same constructs were expressed and purified by a professional production service using the HEK293 expression system and **Ni-NTA affinity chromatography, all three designed scFv variants** were successfully obtained from the culture supernatant. This result suggests that the lack of secretion in our HEK293T setup was primarily due to **expression system limitations** — such as the lower secretion efficiency of HEK293T cells, **and premature signal peptide cleavage**, rather than intrinsic sequence defects.

Together, these findings indicate that all five scFv constructs are in principle capable of secretion under optimized expression conditions, and that differences in processing efficiency between HEK293T and HEK293 systems may critically affect successful protein export.

![](https://static.igem.wiki/teams/5898/notebook/results-img2.webp)

**Figure 6. No detectable scFv expression was observed in the culture supernatant.**

![](https://static.igem.wiki/teams/5898/results/result-figure-7.avif)

**Figure 7. Dot blot verification of anti-IL-23 scFv secretion by HEK293 cells (data from Leadgene Biomedical Inc.)**

## Confirmation of scFv-IL23 Binding

To assess whether our scFv constructs retained their ability to recognize IL-23, we conducted a series of ELISA experiments using three different sample types: (1) concentrated culture supernatants, (2) cell lysates, and (3) purified scFv proteins expressed by a commercial HEK293 system. In each assay, wells were coated with 5 ng of recombinant IL-23 per well as the capture antigen, following the protocol described in the [Experiments – ELISA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) section.

1. Supernatant-based ELISA

   Using concentrated supernatants from our in-house HEK293T transfection, ELISA assays showed that **only the two scFv (p19, p40) constructs derived from clinically used antibody drugs displayed strong binding to IL-23** across two independent batches. In contrast, the three originally designed prototypes showed detectable signals only at the highest concentrations, indicating that these constructs may have low binding affinity to IL-23 or limited protein stability and expression efficiency.

   ![](https://static.igem.wiki/teams/5898/results/result-figure-8.avif)

   **Figure 8. Validation of the affinity of scFvs collected from culture supernatants to IL-23.**
2. Lysate-based ELISA

   We next examined whether the intracellular scFv retained binding activity by performing ELISA with cell lysates. Due to protein loss caused by freeze–thaw treatment, only the first batch yielded usable samples. The results again showed **binding exclusively for the drug-derived scFv constructs**, consistent with the supernatant-based findings.

   ![](https://static.igem.wiki/teams/5898/results/result-figure-9.avif)

   **Figure 9.ELISA validation of the binding capability of scFvs collected from cell lysates.**
3. Purified scFv from commercial HEK293 expression

   To further confirm the binding capability of our original scFv prototypes, we tested  **purified scFv1, scFv2, and scFv3** obtained from a professional HEK293 expression service using **Ni-NTA affinity chromatography**. At the highest tested concentration, **scFv1 and scFv3 exhibited clear IL-23 binding signals**, while **scFv2 did not show a significant difference**, likely due to its relatively lower concentration in the purified sample. These results demonstrate that at least two of our prototype scFv constructs can specifically recognize IL-23 once correctly expressed and processed.

   ![](https://static.igem.wiki/teams/5898/results/result-figure-10.avif)

   **Figure 10. ELISA validation of the binding activity of purified scFv1, scFv2, and scFv3 expressed in HEK293 cells.**

   Together, these findings indicate that while our in-house HEK293T system produced detectable binding only for the drug-derived constructs, the purified scFv expressed by the HEK293 system confirmed that **scFv1 and scFv3 from our original designs also retain IL-23-binding capability**, validating our design concept.

## Functional Validation

To determine the optimal conditions for downstream signaling analysis, we first examined the time-dependent activation of STAT3 in Caco-2 cells following IL-23 stimulation. Cells were treated with 20 ng/mL IL-23 for 15, 30, 60, and 180 minutes, and phosphorylation of STAT3 was analyzed by Western blot. p-STAT3 and p-JAK2 levels both increased at **30 minutes.** Based on this result, the 30-minute timepoint was selected for subsequent co-treatment experiments.

![](https://static.igem.wiki/teams/5898/results/result-figure-13.avif)

**Figure 11. Determination of optimal timepoint for IL-23 stimulation in CaCo2 cells.**

To evaluate the functional effects of anti-IL-23 scFvs on downstream inflammatory signaling, we performed Western blot analysis on Caco-2 cells stimulated with IL-23 and co-treated with individual scFvs for 30 minutes. IL-23 stimulation alone led to a clear increase in p-STAT3 levels, confirming effective activation of the STAT3 pathway.

Among the tested scFvs, only p19 showed a partial inhibitory effect—p-STAT3 expression was reduced to near-baseline levels (1.02 relative to control). scFv #1 produced minimal suppression, with p-STAT3 levels similar to the IL-23–only group, indicating weak or insufficient activity. In contrast, p40 failed to inhibit signaling, raising the possibility of a crosslinking effect or unintended stabilization of the IL-23 receptor complex. Taken together, these results suggest that only **p19 exhibits a measurable inhibitory effect on IL-23–induced inflammatory signaling**, while the other scFvs either lack functional activity or may even enhance pathway activation.

![](https://static.igem.wiki/teams/5898/notebook/new-fig-14.avif)

**Figure 12. Functional validation of anti-IL-23 scFvs (p19, p40. #1) in Caco-2 cells stimulated with IL-23 for 30 min.**

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