Single 3'-exonuclease-based multifragment DNA assembly method (SENAX).

DNA assembly is a vital process in biotechnology and synthetic biology research, during which DNA plasmids are designed and constructed using bioparts to engineer microorganisms for a wide range of applications. Here, we present an enzymatic homology-based DNA assembly method, SENAX (Stellar ExoNuclease Assembly miX), that can efficiently assemble multiple DNA fragments at ambient temperature from 30 to 37 °C and requires homology overlap as short as 12-18 base pairs. SENAX relies only on a 3'-5' exonuclease, XthA (ExoIII), followed by Escherichia coli transformation, enabling easy scaling up and optimization. Importantly, SENAX can efficiently assemble short fragments down to 70 bp into a vector, overcoming a key shortcoming of existing commonly used homology-based technologies. To the best of our knowledge, this has not been reported elsewhere using homology-based methods. This advantage leads us to develop a framework to perform DNA assembly in a more modular manner using reusable promoter-RBS short fragments, simplifying the construction process and reducing the cost of DNA synthesis. This approach enables commonly used short bioparts (e.g., promoter, RBS, insulator, terminator) to be reused by the direct assembly of these parts into intermediate constructs. SENAX represents a novel accurate, highly efficient, and automation-friendly DNA assembly method.