Monthly Progress Report: Enzymatic DNA Synthesis on EWOD Microfluidic Platforms ( Project presentation, Month 1 May-June, 2026)

Program: Program: Primordia Grant

Principal Investigator: David J. Castillo Cornejo PhD

Affiliation: Glyxon Biolabs

Background

Introduction and Project Objectives

The democratization of synthetic biology and the advancement of personalized medicine critically depend on our ability to write de novo genetic code in a rapid, decentralized manner, free of toxic byproducts. Traditional chemical oligonucleotide synthesis (the phosphoramidite method) presents severe limitations, including the use of hazardous organic solvents, inherent degradation of long nucleotide chains due to acidic deprotection steps, and a centralized infrastructure that acts as a bottleneck for global research (Perkel, 2019).

To overcome these limitations, this project proposes the development of an automated, de novo enzymatic DNA synthesis system driven by digital fluid manipulation. The core objective is the engineering and characterization of a coupled system utilizing the template-independent polymerase Terminal Deoxynucleotidyl Transferase (TdT) fused to the silica-binding peptide Car9 (TdT-Car9). This approach aims to optimize the controlled incorporation of nucleotides using immobilized primers coupled with real-time kinetic monitoring.

graph TD
    %% Style Definitions
    classDef default fill:#f9f9f9,stroke:#333,stroke-width:1px,color:#333;
    classDef highlight fill:#eef2ff,stroke:#4f46e5,stroke-width:2px,color:#1e1b4b;
    classDef success fill:#f0fdf4,stroke:#16a34a,stroke-width:2px,color:#14532d;
    classDef warning fill:#fff7ed,stroke:#ea580c,stroke-width:2px,color:#7c2d12;

    %% Main Flow Nodes
    A["💻 <b>Digital Design / Sequence</b><br><br>• Target DNA sequence definition<br><br>• Automated dNTP addition order<br><br>• Microfluidic protocol generation"] --> B
    
    B["🎛️ <b>EWOD Control</b><br><br>• Square electrode matrix actuation<br><br>• Nanoliter droplet manipulation<br><br>• Programmable mixing & washing"] --> C1
    
    subgraph QC ["Biochemical Validation Loop"]
        C2["🧬 <b>Expression Verification</b><br><br>• SDS-PAGE structural check<br><br>• Identity via Western Blot"] --> C3
        C3["🐭 <b>BALB/c OlaHsd Immunization</b><br><br>• TdT injection + CFA/IFA protocol<br><br>• Polyclonal serum collection & verification"] --> C4
        C4["🧪 <b>Stoichiometry Profiling</b><br><br>• Direct/Indirect ELISA assays<br><br>• Quantitative bead loading check<br><br>• Determine: pmol TdT / mg Bead"]
    end
    
    C4 --> C1
    
    C1["🧲 <b>Enzymatic Reaction & Loop</b><br><br>• Reversible enzyme-silica anchoring<br><br>• 5'-Biotin-TEG primer elongation<br><br>• On-chip solid-phase synthesis"] --> D
    
    D["📊 <b>Colorimetric Validation</b><br><br>• H+ ion release in low-buffer system<br><br>• Phenol red spectral shift (Red ➔ Yellow)<br><br>• Real-time kinetics via Bio-Rad 680 (OD560)"]

    %% Assigning Styles
    class A,B highlight;
    class C1,D success;
    class C2,C3,C4 warning;

Fig 1.0- Project Workflow

Methodological Approach (Project Implementation)

The project is divided into key converging technological tracks focused on automation, custom molecular chasses, and frugal science:

1. Expression Vector and Recombinant Production: For the enzymatic machinery, the murine isoform 2 of Terminal Deoxynucleotidyl Transferase was selected due to its structural stability and polymerization kinetics. The codon-optimized sequence fused to the Car9 purification and anchoring tag has been successfully synthesized by Twist Bioscience into a pET28(+) expression vector (pET28+ 6xHis-Tdt-Car9).

This construct has recently been delivered to our logistics broker in San Diego, California, to initiate immediate customs clearance and transit to our facilities for heterologous expression in E. coli.