Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation.

This article focuses on the development of a novel method for constructing highly diverse T7 bacteriophage (phage) libraries displaying random cyclic peptides for peptide screening applications. The authors combined seamless cloning technology, specifically NEBuilder HiFi DNA assembly, with an in vitro cell-free translation system (myTXTL) to overcome challenges associated with the large 36 kb linear T7 phage genome and limitations of conventional packaging extracts. The resulting libraries (CX7C, CX9C, CX11C, and CX13C) demonstrated diversity exceeding 10^9 plaque-forming units with minimal sequence bias, as confirmed by next-generation sequencing. Validation using an anti-FLAG monoclonal antibody successfully enriched the expected epitope sequence, indicating the system's utility for epitope screening. This approach simplifies T7 phage library construction while enhancing diversity and efficiency compared to traditional methods.