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

This project characterizes a series of engineered plasmid constructs designed to enable reliable Gibson Assembly of highly repetitive DNA sequences. Using a synthetic biology framework that combines modular plasmid architecture, insulated regulatory elements, and long-read sequencing validation, we constructed elastin-like polypeptide (ELP) repeat libraries spanning more than three orders of magnitude in length.Each plasmid in this project shares a common modular backbone that includes: (i) a kanamycin resistance cassette with its own promoter and ribosome binding site, (ii) an arabinose-responsive sensor module containing araC and the araBAD promoter, (iii) insulator sequences and transcriptional terminators to separate functional modules, and (iv) a reporter module in which an sfGFP coding sequence and/or ELP repeat cassette is inserted between HindIII and BamHI sites. The ELP region is built from GVGVP-based repeat units, with constructs ranging from 1x to more than 1000x repeats, including both sfGFP-ELP fusion proteins and ELP-only constructs.To validate the integrity and length of these highly repetitive regions, we performed Oxford Nanopore sequencing of the plasmids, supplemented by Sanger sequencing of key junctions and shorter constructs. The long-read data confirm that the modular plasmid design supports robust assembly and maintenance of large repetitive inserts that are difficult to verify using only short-read sequencing or Sanger sequencing alone.This project provides: (1) annotated plasmid sequences deposited in GenBank, (2) raw Nanopore sequencing reads deposited in the SRA, and (3) a generalizable synthetic biology framework for constructing and validating large, repetitive DNA cassettes.