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.

Design

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.

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.

   

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.

   

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.

   

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.

   

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.

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.

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]

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.

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.

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.

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.

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

Engineering Design

ATRA biosynthetic pathway

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

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

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

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

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

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.