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

Abstract

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.

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

“…The parameters optimized for XE cocktail are summarized in Table 1. These results are consistent with data reported for SLiCE and ExoIII cloning by other groups (Dao et al, 2022;Gibson et al, 2009;Guo et al, 2022;Hsiao, 1993;Messerschmidt et al, 2016;Motohashi, 2015;Okegawa & Motohashi, 2015;Zhang et al, 2012), except for the use of ExoT and the dilution buffer. We finally developed a 2Â mix that assembles two DNA fragments with virtually any sequences in 5 min.…”

“…The careful manipulation of the reaction mixture and the use of organic solvents prevented this protocol from being accepted by a wide variety of users. Several groups subsequently reproduced and/or improved the use of ExoIII in the in vitro DNA cloning protocol (Dao et al, 2022; Gibson et al, 2009; Yang et al, 2021). The findings obtained suggested that ExoIII was not only required, but also sufficient for efficient seamless DNA cloning by SLiCE.…”

“…Since the ExoIII enzyme purchased (2170A from Takara‐Bio) contains 25 μL of 200 U/μL enzyme, theoretically, a 10‐μL reaction may be performed ~800,000 times. The use of the commercial product of ExoIII (M0206L from NEB) in the seamless DNA cloning assay was previously reported (Dao et al, 2022). The findings obtained showed that a 30‐fold dilution was optimal for the purchased enzyme.…”

“…Another option is the storage and use of the ExoIII-only solution (X cocktail) and XE cocktail separately to save ExoT when using blunt-ended fragments or those with 5 0 -protruding ends. The protocol for the preparation of recombinant ExoIII was previously reported (Dao et al, 2022). The preparation of these enzymes from E. coli is simple because the contamination of any protein(s) from the E. coli-soluble fraction to the enzymes does not interfere with recombination unless they do not exceed the concentration in SLiCE.…”

“…subsequent studies described re-examinations of and improvements to the protocol (Dao et al, 2022;Gibson et al, 2009;Yang et al, 2021). These modified protocols and that described in this study use the same enzyme, ExoIII, a dsDNA-dependent 3 0 -5 0 exonuclease.…”