iGEM 2025 — Team syphu-china

{% extends "layout.html" %}
{% block title %}Description{% endblock %}
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![hero](https://static.igem.wiki/teams/5630/art/notebook.avif)

* [Notebook](#Notebook)
* [Timeline](#Weeks)
* [Experiments](#Experiments)
* [Materials](#Materials)
* [PDFs](#NotebookPDFs)
* [Data](#DataLinks)
* [Notes](#Notes)

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## Laboratory Notebook

---

A concise, publication-ready record of our wet lab progress across the iGEM season — aligned to the same visual system as *Description*.

**How to use:** The weekly timeline below is filterable. Full Notebook PDFs are embedded openly on this page (no collapses), and each also has an “Open in new tab” link.

Jump to

* [Timeline](#Weeks)
* [Experiments](#Experiments)
* [Materials](#Materials)
* [Notebook PDFs](#NotebookPDFs)
* [Data Links](#DataLinks)
* [Version Notes](#Notes)

## I. Weekly Timeline

---

**Quick filter:**

All
Planning
Molecular
Cell
Analysis
Safety

**Week 1 · Project Kickoff & Ideation**
Mar 10–16, 2025
planning

**Highlights.** Finalized research scope; assigned roles; drafted safety checklist for BSL-1/2 use.

| Item | Owner | Status |
| --- | --- | --- |
| Risk assessment (ATRA handling) | Safety group | Completed |
| Core protocol list (PCR / gel / transformation) | Wetlab | Completed |

**Week 2 · Safety Training & SOP Finalization**
Apr 1–7, 2025
molecular · safety

Completed biosafety training and waste disposal SOP. Bench-tested PCR and gel conditions.

* BSL-1/2 refreshers (spill, sharps, fire).
* Validated PCR annealing temperature grid (56–62 °C).

**Week 3 · First Molecular Runs**
Apr 18–24, 2025
molecular

DNA extraction → PCR → gel verification. Clear bands at expected sizes; purity A260/A280 ≈ 1.9.

**Note.** Kept 10% extra ligation volume to compensate for column loss.

**Week 4 · Plasmid Construction & Transformation**
May 5–11, 2025
molecular · cell

Backbone digestion (EcoRI/XhoI), insert ligation, heat-shock into *E. coli*.

| Sample | Transform. Efficiency | Colony Count | Success |
| --- | --- | --- | --- |
| Empty vector | 1.2×10⁶ CFU/μg | 120 | 100% |
| With insert | 8.5×10⁵ CFU/μg | 85 | 85% |

**Week 5 · Expression Optimization & Quant**
May 18–24, 2025
analysis

IPTG gradient (0.1/0.5/1.0 mM, 4 h) — balanced yield/solubility at 0.5 mM.

| IPTG (mM) | Time (h) | Yield (mg/L) | Purity (%) | Solubility |
| --- | --- | --- | --- | --- |
| 0.1 | 4 | 25.3 | 85 | High |
| 0.5 | 4 | 42.7 | 78 | Medium |
| 1.0 | 4 | 51.2 | 65 | Low |

## II. Detailed Experiments

---

### 1) Plasmid Construction & Transformation

**Objective.** Construct the vector with target insert and transform into *E. coli* for expression.

1. Digest backbone (EcoRI/XhoI)
2. PCR insert, gel purify
3. T4 ligation → competent cells → plate on selective media

### 2) Protein Expression Optimization

**Objective.** Maximize yield while keeping acceptable purity/solubility.

**Conclusion.** 0.5 mM IPTG for 4 h gave best balance.

| Parameter | Mean | SD | N | p-value |
| --- | --- | --- | --- | --- |
| Expression Level (mg/L) | 45.2 | 5.8 | 15 | 0.003 |
| Cell Density (OD600) | 0.85 | 0.12 | 20 | 0.021 |
| Enzyme Activity (%) | 78.3 | 6.5 | 12 | 0.001 |
| Transformation Efficiency | 9.8×10⁵ | 1.2×10⁵ | 8 | 0.045 |

## III. Materials & Reagents

---

| Category | Name | Spec/Notes | Storage |
| --- | --- | --- | --- |
| Plasmid | Backbone pXXX | EcoRI/XhoI | -20 °C |
| Strain | *E. coli* DH5α | chemically competent | -80 °C |
| Antibiotic | Kan / Amp | working 50–100 μg/mL | 4 °C |
| Inducer | IPTG | 1 M stock | -20 °C |

## IV. Notebook PDFs

---

**Direct links:**
[Notebook 1](https://static.igem.wiki/teams/5630/notebook/notebook-1.pdf) ·
[Notebook 2](https://static.igem.wiki/teams/5630/notebook/notebook-2.pdf)
[Notebook (English Translation)](https://static.igem.wiki/teams/5630/notebook/2025-syphu-china-igem-experiment-log-translation.pdf)

### Notebook 1 — inline view

Inline PDF is not supported by your browser.
[Open Notebook 1 in new tab](https://static.igem.wiki/teams/5630/notebook/notebook-1.pdf).

### Notebook 2 — inline view

Inline PDF is not supported by your browser.
[Open Notebook 2 in new tab](https://static.igem.wiki/teams/5630/notebook/notebook-2.pdf).

### Notebook (English Translation) — inline view

Inline PDF is not supported by your browser.
[Open the English-translated Notebook in a new tab](https://static.igem.wiki/teams/5630/notebook/2025-syphu-china-igem-experiment-log-translation.pdf).

## V. Version Notes

---

* v0.1 — Initialized Notebook page following *Description* structure (headings, tables, callouts).
* v0.2 — Added quick filter & DOI copy.
* v0.3 — PDFs displayed directly inline without collapses.

{% extends "layout.html" %}
{% block title %}Description{% endblock %}
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![hero](https://static.igem.wiki/teams/5630/art/engineering.avif)

* [Overview](#eng-overview)
* [Design](#eng-design)
* [Build](#eng-build)
* [Test](#eng-test)
* [Learn](#eng-learn)
* [ATRA – Total Synthesis](#atra-total)
* [References](#References)

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## **Overview**

We have designed a promising microbial therapy that can effectively kill liver cancer cells. We inserted the lactic acid chemotaxis element **IIdR** (containing four lactic acid operon binding sites) into the genome of *E. coli* Nissle 1917 and verified it. Then, we transferred it into two core plasmids, `pET-21a-trc-raldh-IIdR-blh` and `pET-21a-trc-crtEBIY`, for the total synthesis of ATRA. In order to comprehensively and effectively promote the development of this therapy, we follow the engineering cycle recommended by iGEM at every stage of our work. Each engineering cycle consists of four interrelated and sequential steps: design, construction, testing, and learning. In many cases, we have been able to effectively solve the difficulties we encountered and achieve the results we initially expected for our design.

EcN genome edit
IIdR ×4 sites
ATRA total synthesis
`pET-21a-trc-raldh-IIdR-blh`
`pET-21a-trc-crtEBIY`

## **Design**

![Helicobacter pylori, the natural source of TlpC chemoreceptor that mediates lactic acid chemotaxis in gastric colonization](https://static.igem.wiki/teams/5630/engineering/helicobacter-pylori-the-natural-source-of-tlpc-chemoreceptor-that-mediates-lactic-acid-chemotaxis-in-gastric-colonization.webp)

**Figure D-1.** *Helicobacter pylori*, the natural source of TlpC chemoreceptor that mediates lactic-acid chemotaxis in gastric colonization.

**TlpC** is a chemotaxis receptor located on the surface of *Helicobacter pylori* that mediates attraction toward lactic acid and is associated with gastric colonization. We used NCBI-BLAST to identify the closest *E. coli* counterpart to TlpC and designed a chimeric receptor by recombining **TlpC** with an *E. coli* cytoplasmic signaling domain to create a lactic-acid chemoreceptor usable in *E. coli*.

**eTlpC** is a fusion protein comprising the ligand-binding (periplasmic) domain from TlpC and the cytoplasmic signaling domain from *E. coli* strain CFT073. This allows the receptor that normally functions in *H. pylori* to signal in *Escherichia coli*, enabling **EcN** to acquire lactic-acid chemotaxis.

![eTlpC schematic: TlpC ligand-binding domain + CFT073 cytoplasmic signaling domain](https://static.igem.wiki/teams/5630/engineering/etlcp.webp)

**Figure D-2.** eTlpC chimera (TlpC ligand-binding domain + CFT073 cytoplasmic signaling domain) enabling lactic-acid chemotaxis in EcN.

![zoomed]()

## **Build**

1. We use ***E. coli* Nissle 1917** as the template for colony PCR. Using primers
   `OmpT-up-F'` / `OmpT-down-R'`, we amplify the fragment containing **OmpT** and its
   upstream and downstream homology arms.

   ![SDS-PAGE analysis confirming eTlpC protein expression (∼65 kDa) in engineered strains. Lane M: Protein marker, Lane 1: Wild-type EcN, Lane 2: Engineered E3 strain](https://static.igem.wiki/teams/5630/engineering/sds-page-analysis-confirming-etlpc-protein-expression-65-kda-in-engineered-strains-lane-m-protein-marker-lane-1-wild-type-ecn-lane-2-engineered-e3-strain.webp)

   **Figure B-1.** SDS-PAGE analysis confirming eTlpC expression (~65 kDa) in engineered strains.
   Lane M: protein marker; Lane 1: wild-type EcN; Lane 2: engineered E3 strain.
2. Use **T4 DNA ligase** to ligate the PCR product with the linearized **pMD18-T** vector to obtain the recombinant vector
   **pMD18T-OmpT**.
3. Using **pMD18T-OmpT** as template, perform reverse-PCR with primers
   `OmpT-up-R'` / `OmpT-down-F'` to **linearize** it.

   ![pMD18T-OmpT donor / OmpT locus with flanking homology arms](https://static.igem.wiki/teams/5630/engineering/18t-ompt.webp)

   **Figure B-2.** pMD18T-OmpT donor with OmpT flanking arms (safe-harbor integration locus).
4. Using the constructed **pET-21a-trc-eTlpC** as the template, PCR the expression cassette containing the
   `trc` promoter to obtain the **eTlpC** overexpression cassette to be overexpressed.

   ![Schematic representation of recombinant plasmid pET-21a-trc-raldh-IIdR-blh](https://static.igem.wiki/teams/5630/engineering/engineering-3.webp)

   **Figure B-3.** Schematic of recombinant plasmid `pET-21a-trc-raldh-IIdR-blh` (module reference for expression cassettes).
5. Use a **C115 homologous recombination** kit/enzyme to recombine the linearized recombinant vector and the
   **eTlpC** expression cassette for homology recombination, yielding **pMD18T-Donor** that carries the eTlpC cassette flanked
   by OmpT upstream/downstream homology arms for site-specific integration.

   ![Engineering workflow overview for homology recombination and donor assembly](https://static.igem.wiki/teams/5630/engineering/engineering.webp)

   **Figure B-4.** Overview of the engineering workflow for homologous recombination and donor assembly.
6. Amplify the final donor fragment for HDR using primers `OmpT-up-F'` / `OmpT-down-R'`.

To knock **eTlpC** into the EcN genome, we designed a **20-bp sgRNA** targeting the **OmpT** locus and prepared
upstream/downstream homology arms at that site, and constructed the **pEcgRNA-OmpT-N20** plasmid by a **fusion-PCR** method.

![pEcgRNA-OmpT-N20 plasmid constructed by fusion PCR](https://static.igem.wiki/teams/5630/engineering/pecgrna.webp)

**Figure B-5.** `pEcgRNA-OmpT-N20` (fusion-PCR) providing the sgRNA for CRISPR-assisted HDR at the OmpT locus.

![zoomed]()

## **Test**

Transform the **pET-21a-trc-eTlpC** construct into competent **TOP10** cells, pick a transformant colony for PCR,
and verify the correctness through agarose gel electrophoresis.

![Colony PCR verification of eTlpC genomic integration. Lane 1: Wild-type EcN (2223 bp), Lane 2: Engineered E3 strain (3552 bp), confirming successful gene integration.](https://static.igem.wiki/teams/5630/engineering/colony-pcr-verification-of-etlpc-genomic-integration-lane-1-wild-type-ecn-2223-bp-lane-2-engineered-e3-strain-3552-bp-confirming-successful-gene-integration.webp)

**Figure T-1.** Colony PCR verification of **eTlpC** genomic integration. Lane 1: Wild-type EcN (2223 bp);
Lane 2: Engineered E3 strain (3552 bp), confirming successful gene integration.

![zoomed]()

## **Learn**

To ensure the accuracy of plasmid construction and strain identification in our experiments, we verified each part by
**PCR** and **sequencing** upon completion. In addition, we performed a large number of repeated experiments to ensure the
reliability of the results. During plasmid construction, **batch operation** helped maintain the concentration of gene
fragments and improve assembly consistency. To increase the lactic-acid chemotaxis performance of *Escherichia coli*,
while ensuring safety and maximizing its probiotic function during treatment, we plan to make certain modifications to the
chassis cells.

Specifically, the native chemotaxis protein from the **CFT073** system (**GenBank: AAN80760.1**) will be deleted using
**CRISPR-Cas9**, and a plasmid expressing **eTlpC** will be introduced. Given the high concentration of lactic acid in
the cancer microenvironment, this strategy is intended to ensure maximum safety and enhance the colonization ability of the
engineered strain.

# **Total Synthesis of All-trans Retinoic Acid (ATRA)**

## **Design**

The total synthesis route was divided into two parts. The upstream synthesis route used IPP, an endogenous product of
*Escherichia coli*, as the substrate to synthesize the precursor of β-carotene. The downstream synthesis route used
β-carotene as the precursor to synthesize ATRA. The upstream biosynthetic pathway is known from literature. In higher
plants, carotenoids are produced from isopentenyl diphosphate (IPP), which is synthesized in plastids via the mevalonate
pathway (Fig. 1). Under the action of IPP isomerase (IPI) and geranyl diphosphate synthase (GGPS), four IPP molecules
are converted into geranyl diphosphate (GGPP, C20). Two molecules of GGPP are condensed by phytoene synthase
(CrtB in bacteria) to form 15-cis-phytoene, the first specialized compound in the carotenoid pathway. Lycopene, a red
carotenoid found in tomato and watermelon fruits, arises via desaturation by the bacterial phytoene desaturase **crtI**. [1]

![Schematic highlighting the bacterial phytoene desaturase crtI step toward lycopene (engineering-2)](https://static.igem.wiki/teams/5630/engineering/engineering-2.webp)

**Figure ATRA-1.** Bacterial phytoene desaturase **crtI** step toward lycopene (engineering-2).

To obtain the **crtEBIY** gene for use in the project, we collaborated with Ocean University of China to obtain the
`pTrc99a-crtEBIYZ` plasmid, design primers, and amplify the target gene through PCR.

![crtEBIY gene cluster organization from Pantoea ananatis, optimized for expression in E. coli with CAI 0.85](https://static.igem.wiki/teams/5630/engineering/crtebiy-gene-cluster-organization-from-pantoea-ananatis-optimized-for-expression-in-e-coli-with-codon-adaptation-index-cai-of-0-85.webp)

**Figure ATRA-2.** **crtEBIY** cluster (from *Pantoea ananatis*) optimized for expression in *E. coli* (CAI ≈ 0.85).

Through literature review, we learned that in biological systems, retinoic acid is biosynthesized from β-carotene through two
reaction steps (Figure 2). β-carotene is symmetrically cleaved by β-carotene 15,15-oxygenase (**blh**) to generate
retinaldehyde, which is then oxidized by retinal dehydrogenase (**RALDH**) to form retinoic acid. [2] We constructed a total
synthesis pathway for ATRA.

![Comprehensive ATRA biosynthetic pathway engineered in E. coli Nissle 1917: IPP → β-carotene (crtEBIY) → retinal (blh) → ATRA (RALDH)](https://static.igem.wiki/teams/5630/engineering/comprehensive-atra-biosynthetic-pathway-engineered-in-e-coli-nissle-1917-the-pathway-converts-endogenous-isopentenyl-pyrophosphate-ipp-to-carotene-via-crtebiy-enzymes-follow.webp)

**Figure ATRA-3.** End-to-end ATRA pathway in EcN (IPP → β-carotene via **crtEBIY**; β-carotene → retinal via **blh**; retinal → ATRA via **RALDH**).

## **Build**

After confirming the gene sequence, we cloned the target gene into `pET-21a-trc` by homologous recombination and
constructed two core plasmids: `pET-21a-trc-raldh-IIdR-blh` and `pET-21a-trc-crtEBIY`. To enhance gene expression,
we chose the **T7** promoter as the promoter. The **RALDH**, **IIdR**, and **blh** genes were synthesized by GenScript.

![pET-21a-trc-crtEBIY plasmid schematic](https://static.igem.wiki/teams/5630/engineering/21a-crtebiy.webp)

**Figure ATRA-4A.** Plasmid map: `pET-21a-trc-crtEBIY`.

![pET-21a-trc-raldh-IIdR-blh plasmid schematic](https://static.igem.wiki/teams/5630/engineering/21a-raldh-iidr-blh.webp)

**Figure ATRA-4B.** Plasmid map: `pET-21a-trc-raldh-IIdR-blh`.

## **Test**

Transform the constructs containing `pET-21a-trc-raldh-IIdR-blh` and `pET-21a-trc-crtEBIY` into competent
**DH5α** cells, inoculate onto LB agar plates, select the transformed plates using **Amp** antibiotic, and observe the colonies.

![Downstream plasmid transformation: typical efficiency (~1e7 CFU/μg DNA). White colonies indicate no carotenoid production in this module.](https://static.igem.wiki/teams/5630/engineering/downstream-plasmid-transformation-with-typical-efficiency-of-10-cfu-g-dna-white-colonies-confirm-absence-of-carotenoid-production-in-this-module.webp)

**Figure ATRA-5A.** Downstream transformation (~107 CFU·μg−1 DNA): **white colonies** confirm no carotenoid production.

![Upstream plasmid transformation: typical efficiency (~1e8 CFU/μg DNA). Orange colonies indicate β-carotene production.](https://static.igem.wiki/teams/5630/engineering/upstream-plasmid-transformation-results-showing-high-efficiency-10-cfu-g-dna-on-ampicillin-selection-plates-with-orange-colonies-indicating-carotene-production.webp)

**Figure ATRA-5B.** Upstream transformation (~108 CFU·μg−1 DNA): **orange colonies** indicate β-carotene production.

Next, we picked transformants from the LB plates and designed primers for colony PCR to verify plasmid correctness via
agarose gel electrophoresis.

![Learning from failed constructions: initial incomplete integrations (Lanes 1–3) and subsequent optimization](https://static.igem.wiki/teams/5630/engineering/learning-from-failed-constructions-initial-attempts-showed-incomplete-integration-lanes-1-3-leading-to-optimization-of-homology-arm-design-and-transformation-protocols.webp)

**Figure ATRA-6.** Learning from failed constructions: incomplete integrations (Lanes 1–3) led to homology-arm and protocol optimization.

## **Learn**

From the test results, we found that the one-step **homologous recombination (HR)** approach does not efficiently
connect all target genes to a linearized backbone when **fragments are long** and/or there are **multiple inserts**;
recombination efficiency drops with fragment length and fragment count.

To address this, we designed two complementary strategies:

* **Restriction enzyme digestion + ligation** scheme for **long** or **multi-fragment** assemblies, using
  precise junctions to improve correctness and yield.
* An **improved HR scheme** reserved for **short** or **single-fragment** insertions, optimizing overlap length,
  insert:vector molar ratio, reaction temperature/cycling, and cleanup to raise efficiency.

## **Engineering Design**

ATRA biosynthetic pathway

![ATRA pathway](https://static.igem.wiki/teams/5630/model/model-40.webp)

GGPP → Phytoene → Lycopene → β-Carotene → ATRA with key enzymes (crtE, crtB, crtI, crtY); ODE/kinetics support flux allocation.

Upstream module: IPP → β-carotene (crtE/B/I/Y). Downstream module: β-carotene → ATRA (blh, RALDH).
The pathway is split across two plasmids to decouple burden and enable expression tuning.

Pareto front for plasmid optimization

![Pareto front](https://static.igem.wiki/teams/5630/model/model-41-png.webp)

Multi-objective trade-off among GC deviation, CAI, and secondary-structure stability.

A Pareto front is used to balance codon usage (CAI), GC% deviation, and predicted RNA/ORF stability when selecting designs.

Dose–response system curves

![Dose response 1](https://static.igem.wiki/teams/5630/model/model-42-1.webp)

![Dose response 2](https://static.igem.wiki/teams/5630/model/model-42-2.webp)

![EC50 panel 1](https://static.igem.wiki/teams/5630/model/model-43-1.webp)

![EC50 panel 2](https://static.igem.wiki/teams/5630/model/model-43-2.webp)

Hill-type response surfaces with EC50 annotations guide induction and promoter/RBS choices.

Dose–response and EC50 panels define the induction window and help choose appropriate promoters and RBS strengths.

Restriction site density map

![Restriction map](https://static.igem.wiki/teams/5630/model/model-45.webp)

Candidate MCS segments balance cut-site richness and compact length to facilitate cloning strategies.

The multi-cloning site is selected to maximize useful restriction sites while keeping constructs compact and assembly-friendly.

Protein physicochemical properties

![Protein props 1](https://static.igem.wiki/teams/5630/model/model-46.webp)

![Protein props 2](https://static.igem.wiki/teams/5630/model/model-47.webp)

Distributions of molecular weight, pI and instability index help screen stable constructs for expression.

ORF-level physicochemical profiling (MW, pI, instability index) is used to screen constructs for expression stability.

System parameter sensitivity

![Sensitivity heatmap](https://static.igem.wiki/teams/5630/model/model-48.webp)

Global sensitivity map identifies parameters with largest influence on pathway output (priority for tuning).

Sensitivity analysis highlights high-impact parameters/components, providing priorities for tuning and iterative design.

![zoomed]()

## References

---

[1] Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor MA.
Metabolic engineering of carotenoid biosynthesis in plants.
*Trends Biotechnol.* 2008;26(3):139–145.

[2] Han M, Lee P-C.
Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli.
*Microorganisms.* 2021;9(7):1520.

{% extends "layout.html" %}
{% block title %}Description{% endblock %}
{% block page\_content %}

![hero](https://static.igem.wiki/teams/5630/art/experiments.avif)

* [Background](#s1)
* [HCC](#s11)
* [ATRA](#s12)
* [Feasible](#s13)
* [Content](#s2)
* [Pathway](#s21)
* [Strains](#s22)
* [Design](#s3)
* [Basics](#s31)

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## 1. Background

### 1.1 Background and challenges in the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumour and the third leading cause of cancer-related deaths worldwide, with more than 626,000 new cases each year. As a region with high incidence of hepatocellular carcinoma, China has as many as 390,000 deaths due to hepatocellular carcinoma each year, and hepatocellular carcinoma ranks the second most lethal malignant tumour in China. About 7080% of liver cancer patients are already in the middle or late stage when they are found, losing the chance of surgery. Especially in the advanced stage, it is often combined with distant metastases (such as lung, bone, abdominal cavity or lymph nodes, etc.), which makes the treatment very tricky and the prognosis extremely poor.

The strong drug resistance of liver cancer is the main reason limiting the application of chemotherapy. For many years, there has been no breakthrough progress in chemotherapy regimen and efficacy of liver cancer. FOLFOX4 regimen (oxaliplatin + calcium folinate + fluorouracil), as a standard chemotherapy regimen for advanced liver cancer, has an objective remission rate of only 9.1%, and a median survival time of only 10.7 months. Although targeted and immunotherapy have been the first choice for distant metastases in advanced hepatocellular carcinoma in recent years, the efficiency is low and most patients are insensitive to targeted and immunotherapy. Therefore, the development of new strategies that can overcome chemotherapy resistance and improve therapeutic efficacy is an urgent need in the treatment of advanced hepatocellular carcinoma.

### 1.2 ATRA

Alltrans retinoic acid (ATRA), as an active metabolite of vitamin A, was pioneered by a team of Chinese scientists, academician Wang Zhenyi, in the treatment of acute promyelocytic leukaemia (APL) in the 1980s, making APL the first type of leukaemia that can be treated by induced differentiation, and the five-year survival rate of patients increased from less than 20% to more than 90%, creating a miracle in the history of cancer treatment.

With further research, ATRA has also shown potential value in the treatment of many solid tumours.

| Type of cancer | Treatment programme | Mechanism of action | Clinical effectiveness | Level of evidence |
| --- | --- | --- | --- | --- |
| Acute promyelocytic leukaemia | ATRA alone or in combination with arsenic | Induction of differentiation, degradation of PMLRARα fusion protein | 5-year survival rate >90 per cent | Clinical standard treatment |
| pancreatic | ATRA combination chemotherapy | Reversing Vitamin A Deficiency | Median OS extended to 10.9 months | Phase III clinical trials |
| liver cancer | ATRA Joint FOLFOX4 | Induction of differentiation and reduction of chemoresistance | Median OS extended to 16.2 months | Phase III clinical trials |
| Radiotherapy for solid tumours | ATRA combined with radiotherapy | Macrophage polarisation, enhanced T-cell infiltration | 92% reduction in tumour size | Preclinical studies |

### 1.3 Feasibility of ATRA in the treatment of hepatocellular carcinoma

ATRA has diversified mechanisms of action in liver cancer treatment, which can induce the differentiation of tumour stem cells, activate the RAR/RXR signalling pathway, induce TICs to differentiate into mature liver cancer cells and lose their self-renewal ability, and at the same time down-regulate the expression of stem cell markers, such as EpCAM, CD133, and CD90, to improve the efficacy of the FOLFOX regimen; moreover, it has been found that ATRA in combination with chemotherapeutic drugs In addition, it was found that ATRA in combination with chemotherapeutic agents significantly inhibited AKT (Thr308) phosphorylation.Over-activation of the AKT signalling pathway is closely associated with hepatocellular carcinoma progression, drug resistance, and poor prognosis.ATRA, by inhibiting this pathway, reduced tumour cell viability and drug resistance, and promoted chemotherapy-induced apoptosis.

![]()

## 2. Research content

### 2.1 Construction of all-trans retinoic acid total synthesis pathway

Objective: build a de novo biosynthetic route that converts central metabolites into
*all-trans* retinoic acid (ATRA) inside engineered bacteria. The pathway is split into an
upstream carotenoid module and a downstream retinal/retinoic-acid module, enabling stepwise
balancing and orthogonal control.

| Module | Gene(s) | Putative function in the route | Intended product |
| --- | --- | --- | --- |
| Upstream carotenoid module | **crtE, crtB, crtI, crtY** (as **crtEBIY**) | Terpenoid flow to β-carotene via GGPP → phytoene → lycopene → β-carotene | β-carotene |
| Downstream retinal / ATRA module | **blh**, **raldh**, **IIdR** | *blh*: β-carotene 15,15′-dioxygenase (β-carotene → retinal); *raldh*: aldehyde dehydrogenase (retinal → ATRA); *IIdR*: regulatory element to assist expression tuning and pathway balance | Retinal → ATRA |

Design principles: (i) modularization (separate plasmids for upstream and downstream steps to
simplify optimization); (ii) strong-yet-tunable expression (trc promoter–driven cassettes);
(iii) genomic integration (CRISPR–Cas9 assisted) for stability in production strains; and
(iv) assayability through PCR, gel electrophoresis, colony PCR, and downstream metabolite readouts.

#### Planned workflow

1. **Assemble upstream β-carotene synthesis** with the *crtEBIY* cassette to accumulate β-carotene as the direct precursor.
2. **Assemble downstream ATRA synthesis** with *blh* and *raldh*, supported by *IIdR* for expression balance, converting β-carotene → retinal → ATRA.
3. **Balance flux** by adjusting promoter/copy context (plasmid vs. genome) and culture parameters to minimize by-product formation and maximize ATRA.
4. **Verification** by amplification/electrophoresis of each fragment and by metabolite analysis after induction.

Expected outcome: a complete, verifiable ATRA pathway that can be tuned at the module level and
transferred into the final chassis for stable production and downstream testing.

### 2.2 Construction of ATRA engineering bacteria

Objective: create engineered strains that carry the two pathway modules on defined constructs
(plasmid and/or genomic integration) for robust ATRA production and subsequent characterization.

#### Strain and construct strategy

* **Host selection:** laboratory *E. coli* for cloning and amplification; production strain with CRISPR-enabled genomic edits for stability.
* **Upstream plasmid:** pET-21a-trc–**crtEBIY** (β-carotene module).
* **Downstream plasmid:** pET-21a-trc–**raldh-IIdR-blh** (retinal/ATRA module).
* **Genomic integration (as needed):** CRISPR-Cas9–assisted knock-in of the downstream cassette to improve genetic stability and reduce plasmid burden.

#### Key implementation steps

1. **Fragment preparation:** PCR amplification of *crtEBIY*, *blh*, *raldh*, and *IIdR*; agarose gel electrophoresis and gel recovery.
2. **Vector preparation:** pET-21a-trc reverse PCR to open the expression frame (without *lacI*) for homologous recombination.
3. **One-step recombination:** assemble upstream and downstream cassettes using C115 homologous recombinase.
4. **Transformation and screening:** introduce constructs into *E. coli* Top10; select on appropriate antibiotics; verify by colony PCR.
5. **Optional genome editing:** design sgRNA and donor fragments; perform CRISPR-Cas9–mediated integration; cure helper plasmids after verification.
6. **Expression tests:** induce pathway modules, examine β-carotene accumulation and conversion to retinal/ATRA; iterate on promoter strength/copy number if needed.

#### Deliverables

* Validated plasmids: **21a-crtEBIY** and **21a-raldh-IIdR-blh**.
* Confirmed engineered strains for ATRA production (plasmid-borne and/or genome-integrated variants).
* Baseline production data for subsequent optimization in the experimental design section.

## 3. Experimental design

### 3.1 Basic experiments

#### 3.1.1 Preparation of LB medium

**[Experimental apparatus]**
Electronic balance, autoclave, electric stove, medicine spoon, weighing paper, measuring cylinder, beaker, pH test paper, triangular flask, dropper

**[Experimental reagents]**
LB medium formula is shown in the table:
peptone 10 g; yeast powder 5 g; NaCl 10 g; agar 3 g/200 ml; deionised water to 1000 ml

**[Experimental steps]**
1. Accurately calculate and weigh the actual amount of each drug according to the recipe.
2. Use a measuring cylinder to weigh 1000 mL of tap water and pour it into a beaker. Then add each drug (except agar) to the beaker and stir to dissolve.
3. Adjust the pH to 7.2–7.6 with 1 mol/L HCl or 1 mol/L NaOH.
4. Dispense the prepared medium into triangular flasks to no more than 1/2 the height (100 mL of medium for a 250 mL triangular flask, 1.5 g of agar). If preparing solid or semi-solid media, place the weighed agar directly into the triangular flask. Cover with a rubber stopper.
5. Pack the flask with kraft paper, label, and sterilise at 121 °C for 30 min (total cycle ≤1 h 30 min including heat-up/cool-down). Take out the medium promptly after sterilisation and cool it down.
6. Store the sterilised and cooled medium in a cool room.

#### 3.1.2 Plasmid extraction by kit

**[Experimental reagents]**
Plasmid extraction related reagents (Solution I, Solution II, Solution III, Buffer S, Wash Solution, Elution Buffer), anhydrous ethanol, ddH2O

**[Laboratory equipment]**
Pipette tips (blue, yellow), EP tube racks, EP tubes, adsorption columns, small centrifuges, micropipettes, thermostatic water baths, EZ-10 column plasmid extraction kits

**[Experimental procedure]**
1. Collect 1.5–5 ml overnight culture (OD600 ≥ 2.0), spin 12,000 rpm into 1.5 ml tubes, discard medium.
2. Add 250 μl Solution I, fully resuspend. (Ensure RNase A is added when first using Solution I.)
3. Add 250 μl Solution II along the wall, gently invert 5–10×, stand 2 min at RT. (Do not vortex.)
4. Add 250 μl Solution III, gently invert 5–10×. After white floc appears, stand 5 min, spin 12,000 rpm 10 min. Transfer supernatant, add 1/2 vol anhydrous ethanol, invert to mix. (If much starting material, stand 2 min to remove RNA.)
5. Prime EZ-10 Column with 200 μl Buffer S, stand 1 min, spin 8,000 rpm 1 min, discard flow-through. (Use within a day.)
6. Load solution from step 4 (≤750 μl per pass), stand 1 min, spin 8,000 rpm 2 min; repeat until finished.
7. Add 500 μl Wash Solution along the wall, or invert 2–3×, spin 10,000 rpm 1 min.
8. Repeat wash once.
9. Empty and spin 10,000 rpm 2 min to remove residual wash.
10. Air-dry column 10 min at RT or 50 °C 5 min to remove ethanol.
11. Elute with 50 μl Elution Buffer (60 °C), stand 2 min, spin 10,000 rpm 2 min.
12. Store plasmid at −20 °C or use.

#### 3.1.3 PCR

**[Experimental reagents]**
10× PCR buffer, dNTPs, primers, DNA template, Pfu DNA polymerase, ddH2O

**[Laboratory instruments]**
PCR instrument, micropipettes, tips (blue/yellow), EP racks, EP tubes, mini-centrifuge

**[Experimental steps]**
Configure a 20 μl reaction: 10× Buffer (Mg2+) 2 μl; dNTPs 2 μl; each primer 1 μl; template 1 μl; polymerase 0.5 μl; ddH2O 12.5 μl.
Program: 95 °C 5 min; 95 °C 45 s; anneal at Tm 45 s; 72 °C X min (30 cycles); final 72 °C 10 min. (Pfu 600 bp/min; Taq/Ex Taq 1000 bp/min.)
Brief spin; start program.

#### 3.1.4 Agarose gel electrophoresis

**[Experimental apparatus]**
Horizontal electrophoresis, tabletop centrifuge, UV transilluminator, micropipette, Erlenmeyer flask, microwave oven

**[Experimental reagents]**
Ethidium bromide (10 mg/mL), agarose, loading buffer, marker

**[Experimental procedure]**
1. Dissolve 1.0 g agarose in 100 ml 1× TAE by microwaving (use PPE).
2. Cool to 50–60 °C, add 2 μl EB, mix, pour into gel tray with comb (3–5 mm thick). Add 1× TAE to tank (level ≈1 mm above gel).
3. Mix 5 μl PCR product with 1 μl 6× loading buffer, load carefully (new tip each sample).
4. Run 110 V (>40 mA) until bromophenol blue is ~2 cm from the gel front.
5. Image on gel imager (limit UV exposure to protect fragments for gel extraction).

#### 3.1.5 Gel Recovery

**[Experimental apparatus]**
Water bath, balance, high-speed centrifuge, float, 1.5 ml tubes, Thermo GeneJET Gel Extraction Kit (K0692), scalpel

**[Experimental reagents]**
Ethanol 96–100%, isopropanol, 3 M sodium acetate pH 5.2, kit buffers (binding/wash/elution)

**[Experimental steps]**
1. Excise target band with clean blade; minimise UV; record gel weight in pre-weighed tube.
2. Add binding solution 1:1 (v/w) to gel (2:1 if agarose >2%).
3. Dissolve at 50–60 °C for ~10 min; invert intermittently; ensure fully dissolved (yellow indicates optimal pH; if orange/purple add 10 μl 3 M NaOAc).
4. Optional: ≤500 bp add isopropanol 1:2 (v/v); >10 kb add water 1:2 (v/v).
5. Load ≤800 μl per spin to GeneJET column; spin; discard flow-through; repeat until finished (≤1 g agarose/column).
6. Optional: Add 100 μl binding solution; spin; discard.
7. Wash with 700 μl diluted wash; spin 1 min; discard.
8. Spin empty column 1 min to remove ethanol.
9. Elute with 20–50 μl elution buffer (pre-heat to 65 °C for >10 kb). Stand 1 min; spin 1 min.
10. Discard column; store DNA at −20 °C.

#### 3.1.6 Transformation of *E. coli* Top10 by heat-excited receptor cells

**[Experimental reagents]**
LB liquid/solid media, antibiotic, 0.1 M CaCl2, 30% glycerol

**[Experimental instruments]**
Inoculation loop, plate reader, ice box, centrifuges, water bath, incubator, sterile flasks/plates, tubes, micropipettes, tips

**[Experimental steps]**
1. Pick a single colony and culture in LB at 37 °C, 220 rpm for 12–16 h.
2. Inoculate 1–2% into 20 ml LB; 37 °C, 220 rpm to OD620 0.2–0.3.
3. Chill, spin 3000 rpm 10 min on ice.
4. Resuspend in 15–20 ml pre-chilled 0.1 M CaCl2, stand 30 min.
5. Spin; discard supernatant.
6. Resuspend in 1 ml 0.1 M CaCl2 + 1 ml 30% glycerol; aliquot 200 μl; store −20 °C.
7. Thaw on ice; add plasmid; ice 30 min.
8. Heat-shock 42 °C 2 min; ice 3 min.
9. Recover with 800 μl LB at 37 °C for 1–2 h.
10. Spin briefly; plate on selective LB; 37 °C 12–16 h.

#### 3.1.7 Preparation of electroshock receptor states

**[Experimental materials]**
Plates, plasmid, LB, 10% glycerol, antibiotics, tubes, 2 mm electroporation cuvettes

**[Laboratory equipment]**
Incubator, shaker, ice bath, centrifuges, water bath

**[Experimental steps]**
1. Inoculate a single colony into 50 ml LB; 37 °C, 220 rpm overnight.
2. Grow to OD600=0.5; chill on ice ≥15 min.
3. Spin 3000 g, 4 °C, 10 min; discard supernatant.
4. Wash with 30 ml ice-cold 10% glycerol; repeat twice (3000 g, 5 min, 4 °C).
5. Resuspend in 500 μl 10% glycerol; aliquot 100 μl to 1.5 ml tubes.
6. Add 100–200 ng plasmid (~10 μl); ice 10 min; pre-chill cuvette 5 min.
7. Electroporate (2.5 kV, 5 ms; two pulses).
8. Add 900 μl LB; recover 37 °C 1–2 h.
9. Spin 3000 rpm 2 min; resuspend 100 μl LB; plate on selective LB; 37 °C 16 h.

### 3.2 Engineering bacterial construction of downstream plasmid 21a-raldh-IIdR-blh

#### 3.2.1 Amplification and electrophoretic verification of target gene *raldh*, IIdR, *blh* fragments

The amplification method is the same as that described in 3.1.2, and the electrophoresis method is the same as that described in 3.1.4.

**Primer name**   **Primer sequence 5′→3′**
IIdR-F   gaggggttttttgGGAATTCc
IIdR-R   caggacccactagtatgattg
blh-F   ctgggtaaaacaatcatactag
blh-R   gtggtggtggtggtgcTCGAGCGGTT
raldh-F   gtttaactttaagaaggag
raldh-R   ggtgagaatgGAATTCCcaa

[![blh amplification gel](https://static.igem.wiki/teams/5630/experiments/experiments2/blh-amplification-results.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/blh-amplification-results.webp)

**Figure 1.** *blh* amplification results.

[![lldR amplification gel](https://static.igem.wiki/teams/5630/experiments/experiments2/iidr-amplification-results.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/iidr-amplification-results.webp)

**Figure 2.** lldR (IIdR) amplification results.

[![raldh amplification gel](https://static.igem.wiki/teams/5630/experiments/experiments2/raldh-amplification-results.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/raldh-amplification-results.webp)

**Figure 3.** *raldh* amplification results.

#### 3.2.2 Gel recovery of target gene *raldh*, IIdR, *blh* fragments

The gel recovery method is the same as described in 3.1.3.

#### 3.2.3 Kit extraction of pET-21a-trc plasmid

The plasmid extraction method is the same as described in 3.1.1.

#### 3.2.4 Reverse PCR and electrophoretic verification of pET-21a-trc plasmid

**[Purpose of experiment]**
Reverse amplification of pET-21a-trc plasmid to obtain the expression frame without *lacI*, facilitating homologous recombination with the *raldh-IIdR-blh* fragment to construct recombinant plasmid 21a-blh-raldh-IIdR.

**[Experimental materials]**
pET-21a-trc plasmid

**Primer name**   **Primer sequence 5′→3′**
ATRA plasmid anti-p primer 1   TTGAACTTGTCATatgtatatctccttcttaaagttaaacaaa
ATRA anti-p primer 2   CGTTCCGTAACCGCTCGAgcaccaccaccaccaccactg

**[Experimental reagents]**
ddH2O, 10× PCR buffer, dNTP, Pfu DNA polymerase

**[Experimental apparatus]**
Mini-centrifuge, PCR instrument, micropipettes, tips, EP tubes, racks, water bath

**[Experimental procedure]**
1. PCR as in 3.1.1.
2. De-templating system in 1.5 ml tube: ddH2O 7 μl; Q.Cut DpnI 1 μl; 10× Cut Buffer 2 μl; 21a reverse PCR product 0.5 μl.
3. Incubate 37 °C 1 h.
4. Inactivate at 70 °C 15 min.
5. Electrophoresis as in 3.1.4.

[![21a reverse-PCR verification (anti-P)](https://static.igem.wiki/teams/5630/experiments/experiments2/21a-anti-p-verification.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/21a-anti-p-verification.webp)

**Figure 4.** 21a anti-P verification.

Reverse PCR of pET-21a-trc generated the lacI-free expression frame for recombination.

#### 3.2.6 Construction of plasmid 21a-blh-raldh-IIdR using homologous recombination methods

**[Aim of experiment]**
Construct plasmid 21a-raldh-IIdR-blh using C115 homologous recombinase

**Experimental materials**
*raldh*, IIdR, *blh* gene fragments, linearised 21a plasmid fragments

**[Experimental reagents]**
ddH2O, C115 homologous recombinase

**Experimental apparatus**
EP tube, rack, PCR instrument, tips, micropipette, water bath

**Experimental steps**
Configure 30 μl reaction: Linearised 21a 12 μl; blh 1 μl; IIdR 1 μl; raldh 0.8 μl; ddH2O 0.2 μl; ClonExpress Ultra One-Step 15 μl. Incubate 50 °C 30 min.

[![Map of recombinant plasmid 21a-raldh-lldR-blh](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-representation-of-recombinant-plasmid-21a-raldh-iidr-blh.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-representation-of-recombinant-plasmid-21a-raldh-iidr-blh.webp)

**Figure 5.** Schematic of recombinant plasmid 21a-raldh-lldR-blh.

#### 3.2.7 Transformation of Top10 by heat-stimulated sensory state

The recombinant plasmid was transformed into *E. coli* Top10 to amplify the recombinant plasmid for subsequent experiments. Receptor preparation and transformation methods are the same as those shown in 3.1.5.

#### 3.2.8 Colony PCR to verify expression

### 3.3 Engineering bacterial construction of upstream plasmid 21a-crtEBIY

#### 3.3.1 Amplification and electrophoretic verification of the target gene *crtEBIY* fragment

Amplification method as described in 3.1.2, electrophoresis method as described in 3.1.4.

**Primer name**   **Primer sequence 5′→3′**
Target gene primer 1   ctttaagaaggagatataTTCATGACGGTCTGCGC
Target gene primer 2   ggggttatgctagttattgcTTAACGATGAGTCGTCATAATGGC

[![crtEBIY amplification gel](https://static.igem.wiki/teams/5630/experiments/experiments2/crtebiy-amplification-results.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/crtebiy-amplification-results.webp)

**Figure 6.** *crtEBIY* amplification results.

#### 3.3.2 Gel recovery of the target gene *crtEBIY* fragment

The gel recovery method is the same as described in 3.1.3.

#### 3.3.3 Kit extraction of pET-21a-trc plasmid

Same as experiment 3.2.3

#### 3.3.4 Reverse PCR and electrophoretic verification of pTrc99a-crtEBIYZ plasmid

Same as experiment 3.2.4

**Primer name**   **Primer sequence 5′→3′**
β-carotene plasmid primer 1   GCGCAGACCGTCATGAAtatatctccttcttaaag
β-carotene plasmid primer 2   ttatgacgactcatcgttaaGCAATAACTAGCATAACCCCTTGG

#### 3.3.5 Construct plasmid 21a-crtEBIY using homologous recombination method

As in experiment 3.2.6, the recombination system is as follows:

**makings**   **dosage**
Linearised 21a plasmid fragment 2 μl; crtEBIY gene fragment 3 μl; ClonExpress Ultra One-Step 5 μl

[![Map of recombinant plasmid 21a-crtEBIY](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-representation-of-recombinant-plasmid-21a-crtebiy.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-representation-of-recombinant-plasmid-21a-crtebiy.webp)

**Figure 7.** Schematic of recombinant plasmid 21a-crtEBIY.

#### 3.3.6 Heat-stimulated sensory state transformation Top10

Same as experiment 3.2.6

#### 3.3.7 Colony PCR to verify expression

### 3.4 Gene integration of downstream plasmids using the CRISPR-Cas9 system

#### 3.4.1 Design of sgRNA and construction of pEcgRNA plasmid

**[Aim of experiment]**
Design and construction of sgRNA and pEcgRNA plasmids for targeted knockdown of *endA* in the EcN genome, in order to facilitate subsequent integration of the *raldh-IIdR-blh* cassette.

**[Experimental Materials]**
pEcgRNA, BsaI enzyme

**[Experimental reagents]**
ddH2O, G buffer, T4 ligase buffer, LB/Sm plates (50 μg/mL), 5 ml LB+Sm (50 μg/mL)

**[Experimental apparatus]**
Micropipettes, tips, EP tubes, racks, water bath, applicator, biosafety bench, incubator

**Experimental steps**
1. Design sgRNA (20 bp) on the target site with CHOPCHOP: cgtagagtgggaacacgtcg, PAM: CGG.
2. Digest ccdB with BsaI to linearise pEcgRNA (cohesive ends 5′-TAGT / 5′-AAAC). Digest 20 μl: pEcgRNA 3; BsaI 1; G buffer 2; ddH2O 14.
dsDNA (N20) ligation system (50 μl): ddH2O 35; N20-up 5; N20-dn 5; T4 ligase buffer 5.
3. 95 °C 5 min; cool down stepwise; 16 °C 10 min.
4. Dilute dsDNA 200×, ligate 1 μl dsDNA + 1 μl BsaI-linearised pEcgRNA at 16 °C for 1 h with T4 ligase.
5. Transform into Top10, plate on Sm (50 μg/mL), 37 °C overnight; pick colonies into 5 ml LB+Sm and culture 220 rpm 37 °C overnight; extract plasmid and verify by digestion next day.

[![Flowchart of pEcgRNA-N20 construction](https://static.igem.wiki/teams/5630/experiments/experiments2/flowchart-of-pecgrna-n20-plasmid-construction.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/flowchart-of-pecgrna-n20-plasmid-construction.webp)

**Figure 8.** Flowchart of pEcgRNA-N20 plasmid construction.

#### 3.4.2 Fusion PCR to obtain donor destination fragments

**[Purpose of experiment]**
Construction of recombinant vector pMD18T-Donor containing upstream and downstream homology arms of *endA* by fusion PCR method.

**[Experimental materials]**
pMD 18T vector

**Primer name**   **Primer sequence 5′→3′**
endA-up-F   aactctcttacacccagcgc
endA-up-R   tctcgatcctctacggacaaataacggtacatcacttactccg
endA-down-F   atatccggattggcgggcgcgaaagagctaacc
endA-down-R   ttgcaatcttctgccactgct

**[Experimental reagents]**
T4 ligase, ddH2O, 10× ligase buffer

**[Experimental apparatus]**
Micropipette, water bath, balance, 1.5 ml tubes

**[Experimental steps]**
1. Colony PCR on EcN to amplify *endA* with its homology arms (PCR as 3.1.1).
2. Ligate with linearised pMD18-T using T4 ligase → pMD18-T-endA.
3. Use pMD18-T-endA as template; reverse-PCR with endA-up-R′/endA-down-F′ to linearise (method as 3.2.4).
4. Use pET-21a-trc-raldh-IIdR-blh (10× diluted) as template to PCR the trc-promoter expression frame (4318 bp).
5. Electrophoresis to verify the expression frame.
6. Gel recovery.
7. Homologous recombination (C115) between the linearised vector and the r-I-b expression frame → pMD18T-Donor (contains r-I-b and *endA* arms). Finally amplify Donor with endA-up-F′/endA-down-R′ (5289 bp) and verify by electrophoresis.

[![pMD-18T-endA map](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-diagram-of-pmd-18t-enda.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-diagram-of-pmd-18t-enda.webp)

**Figure 9.** Schematic diagram of pMD-18T-endA.

[![pMD-18T-raldh-lldR-raldh (Donor) map](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-diagram-of-pmd-18t-raldh-iidr-raldh.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/schematic-diagram-of-pmd-18t-raldh-iidr-raldh.webp)

**Figure 10.** Schematic diagram of pMD-18T-raldh-IIdR-raldh (Donor).

#### 3.4.4 Preparation of EcN (pEcCas) electroshock receptor cells

The preparation method is the same as described in 3.1.7

#### 3.4.5 Electroshock transformation of DNA fragments with pEcgRNA

The electroporation cuvette was pre-cooled on ice for 5–10 min, then 13 μl of DNA fragment (gene homologous recombination sequence) and 3 μl of pEcgRNA-gene-N20 were added to 200 μl of EcN electrocompetent cells, gently mixed and transferred to a sterile 2 mm cuvette for electroporation (2.5 kV, 5 ms, twice). Add 900 μl drug-free LB, recover 37 °C for 1–2 h, pellet, resuspend in 100 μl LB and plate on LB with Kan (25 μg/mL) + Sm (50 μg/mL), 37 °C overnight. Negative control: cells without L-arabinose induction.

#### 3.4.6 Translator Elimination and Validation

Positive mutant colonies were PCR-checked with gene-up-F/gene-dn-R (wild-type EcN as negative control). Pick positives and culture in LB with 10 mM rhamnose and Kan, 37 °C, 220 rpm overnight; transfer to drug-free LB for 12 h; plate on sucrose LB (3 g/L) to isolate single colonies. Screen on LB, Kan-LB and Sm-LB. Single colonies that fail on Kan and Sm but grow on drug-free plates indicate successful knockout/integration and loss of pEcgRNA-gene-N20 and pEcCas.

### 3.3 Experimental Results

#### 3.3.1 Integration of Lactic Acid-Targeting Protein Gene

*Acquisition and verification of OmpT donor and related fragments*

[![PCR electrophoresis of OmpT (Fragment A)](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-ompt-fragment-a.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-ompt-fragment-a.webp)

**Figure 3-5-1.** PCR electrophoresis of *OmpT* (Fragment A).

[![Colony PCR verification of 18T-OmpT](https://static.igem.wiki/teams/5630/experiments/experiments2/colony-pcr-verification-electrophoresis-diagram-of-18t-ompt.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/colony-pcr-verification-electrophoresis-diagram-of-18t-ompt.webp)

**Figure 3-5-2.** Colony PCR verification electrophoresis of 18T-*OmpT*.

[![Reverse PCR electrophoresis of 18T-OmpT](https://static.igem.wiki/teams/5630/experiments/experiments2/reverse-pcr-electrophoresis-diagram-of-18t-ompt.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/reverse-pcr-electrophoresis-diagram-of-18t-ompt.webp)

**Figure 3-5-3.** Reverse-PCR electrophoresis of 18T-*OmpT*.

[![PCR of 21a-trc-etlpC expression cassette](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-21a-trc-etlpc-expression-cassette.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-21a-trc-etlpc-expression-cassette.webp)

**Figure 3-5-4.** PCR of 21a-trc-*etlpC* expression cassette.

[![PCR of homology arms and etlpC](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-upstream-and-downstream-homology-arms-of-integration-site-and-etlpc-gene.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/pcr-electrophoresis-diagram-of-upstream-and-downstream-homology-arms-of-integration-site-and-etlpc-gene.webp)

**Figure 3-5-5.** PCR electrophoresis of upstream/downstream homology arms and the *etlpC* fragment.

*Verification of etlpC gene integration*

[![Colony PCR verifying etlpC integration](https://static.igem.wiki/teams/5630/experiments/experiments2/colony-pcr-electrophoresis-verification-diagram-of-etlpc-gene-integration.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/colony-pcr-electrophoresis-verification-diagram-of-etlpc-gene-integration.webp)

**Figure 3-5-6.** Colony PCR verification of *etlpC* gene integration (EcN 2223 bp; E3 3552 bp).

*Expression and functional verification*

[![Lactic acid targeting verification of E3 strain](https://static.igem.wiki/teams/5630/experiments/experiments2/verification-of-lactic-acid-targeting-of-e3-strain.webp)](https://static.igem.wiki/teams/5630/experiments/experiments2/verification-of-lactic-acid-targeting-of-e3-strain.webp)

**Figure 3-5-7.** Lactic acid targeting of E3 strain.

Visible turbidity around 0.1 mol/L lactic-acid filter paper; absent near the negative control.

![]()

### Virtual Experiments · Demo Videos

A selection of virtual experiment demonstrations for education and public outreach.

Clip 1

Clip 2

Clip 3

Clip 4

Clip 5

Clip 6

![Online Molecular Biology Experiment Animation Simulation System – platform screenshot](https://static.igem.wiki/teams/5630/education/experiments-platform.webp)

Virtual Experiment Platform

## Online Molecular Biology Experiment Animation Simulation System

Interactive web platform for demonstrating molecular-biology procedures with step-by-step animations.
Suitable for education and public outreach (no lab setup required).

Link:
<https://experiment-platform-ulp2vhzlm7xcpyh3bp2rvw.streamlit.app/>

[Open Platform](https://experiment-platform-ulp2vhzlm7xcpyh3bp2rvw.streamlit.app/)
Copy Link
Show QR

![Platform QR code](https://api.qrserver.com/v1/create-qr-code/?size=180x180&data=https%3A%2F%2Fexperiment-platform-ulp2vhzlm7xcpyh3bp2rvw.streamlit.app%2F)

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{% extends "layout.html" %}
{% block title %}Description{% endblock %}
{% block page\_content %}

![hero](https://static.igem.wiki/teams/5630/art/results.avif)

* [Overview](#results)
* [Summary](#executive-summary)
* [Genes](#gene-amplification)
* [Plasmids](#plasmid-construction)
* [Proteins](#protein-expression)
* [Pathway](#metabolic-pathway)
* [Lactate](#lactate-regulation)
* [Stability](#genetic-stability)
* [Metrics](#key-metrics)
* [Conclusion](#conclusion)

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## Results 2025 iGEM · SYPHU-CHINA

Complete ATRA biosynthetic pathway in *E. coli*: from gene cloning to functional validation.

---

### Executive Summary

**Summary.** We established a complete biosynthetic route for all-trans retinoic acid (ATRA) in *E. coli*, covering PCR amplification, plasmid assembly, protein expression, pathway integration, lactate-responsive regulation, and stability evaluation.

* **Full genetic stack:** all key genes amplified and verified; upstream β-carotene and downstream retinal→ATRA modules constructed and co-expressed.
* **Strong chassis performance:** high transformation efficiency; robust enzyme expression.
* **Functional outputs:** β-carotene (upstream) and ATRA (combined strain) detectable.
* **Smart control:** lactate-responsive induction with wide dynamic range in a physiologically relevant window.
* **Operability:** plasmid retention >90% over 20 generations under our conditions.

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### Gene Amplification and Verification

**Goal.** Obtain clean, size-correct PCR products to maximize downstream assembly efficiency.

![Gene Amplification Analysis](https://static.igem.wiki/teams/5630/results/gene-amplification-analysis.webp)

**Figure A.** Gene amplification analysis (gene-amplification-analysis). Representative gels and QC summaries for the four pathway genes.

*Interpretation:* Band sizes match theoretical lengths within tolerance; A260/A280 indicates high nucleic-acid purity suitable for cloning.

##### Analytical summary of PCR products

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| Gene | Theoretical size | Experimental size | Concentration (ng/μL) | Purity (A260/A280) | Amplification efficiency (%) |
| --- | --- | --- | --- | --- | --- |
| blhSR | 1,812 bp | 1,809 ± 7 | 45.2 ± 3.1 | 1.87 ± 0.03 | 94.3 ± 2.1 |
| raldhHS | 1,521 bp | 1,518 ± 5 | 38.7 ± 2.8 | 1.85 ± 0.02 | 96.1 ± 1.8 |
| IIdR | 897 bp | 894 ± 4 | 52.1 ± 4.2 | 1.89 ± 0.04 | 97.8 ± 1.2 |
| crtEBIY | 5,214 bp | 5,198 ± 12 | 28.3 ± 2.5 | 1.83 ± 0.05 | 85.4 ± 3.5 |

---

### Plasmid Construction and Verification

**Strategy.** Homologous recombination for upstream (crtEBIY) and downstream (blhSR–raldhHS) modules; validated by colony PCR and sequencing.

![Plasmid Quality Analysis](https://static.igem.wiki/teams/5630/results/plasmid-quality-analysis.webp)

**Figure B.** Plasmid quality analysis (plasmid-quality-analysis). Concentration, purity indices and functional colony screens.

*Interpretation:* High A260/A230 & A260/A280 and >108 CFU/μg transformation support plasmid integrity; positive-clone rate >88% confirms efficient assembly.

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Compact

| Plasmid construct | Conc. (ng/μL) | A260/A280 | A260/A230 | Transform. efficiency (CFU/μg) | Positive clones (%) |
| --- | --- | --- | --- | --- | --- |
| Downstream pathway | 68.5 ± 5.2 | 1.86 ± 0.02 | 2.15 ± 0.05 | (2.8 ± 0.3)×10⁸ | 92 ± 3 |
| Upstream pathway | 72.3 ± 4.8 | 1.84 ± 0.03 | 2.12 ± 0.07 | (2.5 ± 0.2)×10⁸ | 88 ± 4 |
| *Acceptance criteria* | >50 | 1.8–2.0 | >2.0 | >1×10⁸ | >80 |

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### Protein Expression and Functional Characterization

**Result.** SDS-PAGE confirmed robust expression at expected MW. Induction at 25–30 °C balanced total expression and solubility.

![Temperature Optimization Analysis](https://static.igem.wiki/teams/5630/results/temperature-optimization-analysis.webp)

**Figure C.** Temperature optimization (temperature-optimization-analysis). Solubility vs. total expression across induction temperatures.

*Interpretation:* 25–30 °C yields the best compromise for active enzymes supporting downstream flux.

##### Expression & activity metrics

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| Enzyme | Theor. MW (kDa) | Expression | Solubility | Specific activity (U/mg) | Note |
| --- | --- | --- | --- | --- | --- |
| BlhSR | 67.8 | Strong | 68 ± 5% soluble | 12.3 ± 1.2 | Drives retinal formation |
| RALDH | 55.1 | Strong | 75 ± 4% soluble | 8.7 ± 0.9 | Terminal oxidation to ATRA |
| CrtE/B/I/Y | 20–44 | Moderate | Membrane-associated | N/A | β-carotene confirmed |

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### Metabolic Pathway Functionality

**Outcome.** Flux from β-carotene to retinal and ATRA was confirmed using phenotype and LC–MS quantification.

![Static Metabolic Pathway](https://static.igem.wiki/teams/5630/results/static-metabolic-pathway.webp)

**Figure D.** Static metabolic pathway (static-metabolic-pathway). Upstream carotenoid & downstream retinoid modules overview.

*Context:* The schematic maps measured intermediates to specific enzymatic steps.

![Metabolite Production Analysis](https://static.igem.wiki/teams/5630/results/metabolite-production-analysis.webp)

**Figure E.** Metabolite production (metabolite-production-analysis). Module-wise outputs vs full pathway.

*Interpretation:* Retinal >> ATRA implies RALDH oxidation is rate-limiting → priority for enzyme/cofactor engineering.

![Production Kinetics Analysis](https://static.igem.wiki/teams/5630/results/production-kinetics-analysis.webp)

**Figure F.** Production kinetics (production-kinetics-analysis). Growth-associated β-carotene formation across 24 h.

*Interpretation:* Peak volumetric productivity near transition to stationary phase → informs induction timing for fed-batch.

▶ Time-course (24 h) table

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| Time (h) | OD600 | β-Carotene (mg/L) | Vol. productivity (mg·L⁻¹·h⁻¹) | Specific yield (mg/OD) |
| --- | --- | --- | --- | --- |
| 0 | 0.65 ± 0.05 | 0.05 ± 0.01 | – | 0.08 |
| 6 | 1.82 ± 0.08 | 0.38 ± 0.06 | 0.055 | 0.21 |
| 12 | 2.95 ± 0.12 | 1.12 ± 0.15 | 0.123 | 0.38 |
| 18 | 3.42 ± 0.15 | 1.89 ± 0.22 | 0.128 | 0.55 |
| 24 | 3.68 ± 0.18 | 2.35 ± 0.32 | 0.077 | 0.64 |

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### Lactate-Responsive Regulation

**Relevance.** Tumor-like lactate levels (1–10 mM) trigger a strong, selective induction with acceptable viability.

![Lactate Induction Analysis](https://static.igem.wiki/teams/5630/results/lactate-induction-analysis.webp)

**Figure G.** Lactate induction (lactate-induction-analysis). Dose–response across a physiological window.

*Interpretation:* Response window overlaps tumor microenvironment; supports in situ activation concepts.

▶ Temporal activation after induction

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| Time (min) | Protein (%) | mRNA (rel.) | Apparent translation | State |
| --- | --- | --- | --- | --- |
| 0 | 100 | 100 | 1.00 | Baseline |
| 15 | 185 ± 12 | 220 ± 15 | 0.84 | Initial |
| 30 | 420 ± 25 | 580 ± 32 | 0.72 | Accumulation |
| 60 | 720 ± 35 | 850 ± 42 | 0.85 | Near-max |
| 120 | 870 ± 28 | 920 ± 38 | 0.95 | Steady |

---

### Genetic Stability

**Finding.** High plasmid retention and functionality across serial passages; suitable for batch and short continuous runs.

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| Generation | Plasmid retention (%) | Functional expression (%) | Segregational loss (%/gen) | Specific productivity (rel.) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 100 | – | 100 |
| 5 | 98 ± 1 | 97 ± 2 | 0.40 | 97 |
| 10 | 96 ± 2 | 94 ± 3 | 0.41 | 94 |
| 15 | 94 ± 2 | 91 ± 3 | 0.43 | 91 |
| 20 | 92 ± 3 | 88 ± 4 | 0.45 | 88 |

*Interpretation:* For very long continuous processes, consider genomic integration or addiction systems to further reduce segregational loss.

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### Key Performance Indicators

**Visualization set.** Bubble chart for milestone impact, 3D response surface for interaction effects, parallel coordinates for multi-objective trade-offs.

![Performance Metrics Bubble Chart](https://static.igem.wiki/teams/5630/results/static-bubble-chart.webp)

**Figure H.** Performance metrics bubble chart (static-bubble-chart). Compact view of milestones vs relative impact.

*Interpretation:* Bubble size/position highlight key contributors (e.g., cloning efficiency, inducible control range).

![3D Response Surface](https://static.igem.wiki/teams/5630/results/3d-response-surface.webp)

**Figure I.** 3D response surface (3d-response-surface). Output predicted across two interacting factors under fixed constraints.

*Interpretation:* A ridge emerges at moderate induction and mid-temperature, consistent with wet-lab screens.

![Parallel Coordinates Optimization](https://static.igem.wiki/teams/5630/results/parallel-coordinates-optimization.webp)

**Figure J.** Parallel-coordinates optimization (parallel-coordinates-optimization). Feasible regions satisfying yield, stability and viability constraints.

*Interpretation:* Guides DBTL cycles by exposing trade-offs and Pareto-like fronts.

▶ KPI summary table

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| Metric | Target | Achieved | Rating | Significance |
| --- | --- | --- | --- | --- |
| Gene cloning | 100% | 100% | Excellent | Foundation for DBTL cycles |
| Plasmid construction | >80% | 90% | Excellent | Faster iteration |
| Transformation | >10⁸ CFU/μg | 2.8×10⁸ | Exceeded | Library readiness |
| Protein expression | Detectable | Strong | Excellent | Functional capacity |
| β-Carotene | Detectable | 2.35 mg/L | Achieved | Upstream validated |
| ATRA | Detectable | 28.4 nM | Achieved | Full pathway confirmed |
| Induction range | >5-fold | 8.7-fold | Excellent | Precise control |
| Stability (20 gen) | >80% | 92% | Exceeded | Scale-up feasibility |

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### Analysis and Discussion

▶ Technical achievements

* **Complete pathway integration** with detectable ATRA output.
* **High-efficiency cloning & recombination** framework (>90% positive clones).
* **Lactate-responsive circuit** with large dynamic range.
* **Kinetic profiling** informs temperature and induction set-points.

▶ System insights

* **25–30 °C** balances solubility and expression for active yield.
* **RALDH oxidation** appears rate-limiting (retinal accumulation).
* **Low segregational loss** suggests manageable burden across passages.

▶ Translational potential

* **Sustainable ATRA bioproduction** complementing chemical synthesis.
* **Environment-responsive control** for tumor-localized activation concepts.
* **Platform extensibility** to other retinoids.

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### Conclusion

We reconstructed an end-to-end ATRA pathway in *E. coli* and validated each layer—genetics, expression, metabolism, regulation, and stability. While yield optimization remains, these results establish a robust framework for further engineering and application.

*All experiments followed iGEM safety guidelines and institutional biosafety protocols. Complete procedures and raw data are available upon request.*

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