A Synthetic Biology Framework Integrating Modular Plasmid Design, Gibson Assembly, and Nanopore Sequencing for Constructing Highly Repetitive DNA

ABSTRACT: A robust framework for the precise engineering of highly repetitive DNA sequences, a longstanding challenge in synthetic biology due to their resistance to direct synthesis, assembly, and sequence verification. As a proof of concept, we constructed plasmids encoding repeats of the pentapeptide Gly-Val-Gly-Val-Pro (GVGVP)ₙ, which form elastin-like polypeptides (ELPs) with tunable, temperature-dependent solubility. Leveraging the redundancy of the genetic code, a synthetic DNA fragment encoding GVGVP₁₇ was incorporated into a plasmid with a modular restriction-site architecture that enables iterative repeat amplification through a digest-and-assemble workflow. Sequential HindIII and BamHI digestion followed by Gibson Assembly expanded the repeat number (2n-1) while preserving plasmid integrity, yielding constructs up to GVGVP₁₀₂₅ confirmed by nanopore whole-plasmid sequencing. Fusion of superfolder GFP to the GVGVP library enabled expression and functional characterization of ELPs up to 129 repeats, whereas longer sequences exhibited instability in E. coli BL21(DE3). This scalable and precise approach overcomes major barriers in the cloning and verification of repetitive DNA and establishes a versatile platform for constructing large, programmable biomolecules.

ORGANISM(S): Escherichia coli

SUBMITTER: Grace Curtician 

PROVIDER: S-BSST2329 | biostudies-other |

REPOSITORIES: biostudies-other