Oligonucleotide library synthesis using TdT

Recent applications in synthetic biology require an expanded repertoire of bioparts to ensure the reliable function of increasingly complex synthetic systems. Several strategies have been proposed to discover functional bioparts from randomized or synthetic DNA sequences. Among them, the template-independent polymerization ability of terminal deoxynucleotidyl transferase (TdT) has emerged as a versatile approach for randomized oligonucleotide synthesis. However, intrinsic kinetic biases and nucleotide preferences of TdT impose a major constraint on precise control of base composition, which is essential for constructing tailored regulatory elements. Here, we systematically characterized the base-incorporation biases under various nucleotide compositions and demonstrated tunable control over the base composition of TdT-synthesized oligonucleotides by modulating reaction parameters. By screening oligonucleotide libraries optimized for distinct classes of bioparts, we identified a suite of functional bioparts spanning a broad range of expression strengths. Finally, we demonstrate a one-pot workflow that integrates TdT-based oligonucleotide synthesis with metabolic pathway optimization, enabling the rapid assembly and screening of a transcriptionally optimized lycopene biosynthetic pathway in Escherichia coli.