Recent advances in DNA assembly technologies

Abstract

DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods.

Keywords: automation; genome synthesis; metabolic engineering; pathway construction; pathway optimization; synthetic biology.

Figure 1.

Key DNA assembly methods.

Figure 2.

Improved in vivo sequence homology-based DNA assembly methods in Saccharomyces cerevisiae. (a) In Wingler and co-workers' method (Wingler and Cornish, 2011), Type A and Type B are alternated in the series of donor plasmids harboring the fragments to be assembled. The homing endonuclease coded by the donor plasmid specifically cuts the integration site on the genome to promote integration of the next fragment. The markers are reused in turns to guarantee that the corresponding fragments are successfully appended. Mod represents modulo operation. ‘H. Arm’ represents homologous arms. (b) In Kuijpers and co-workers' method (Kuijpers et al. 2013), the yeast replicon and marker fragments on the vector backbone are separated to reduce the likelihood of generating false positives.

Figure 3.

Schematic of three applications based on in vivo DNA assembler method. (a) Promoter libraries are generated by error-prone PCR and assembled in front of each gene in the pathway. (b) For each enzyme in the pathway, homologous genes from various microorganisms are cloned and are flanked by the arms homologous to their neighboring promoters and terminators. All expression cassettes are then assembled using the DNA assembler method. (c) All enzymes in the pathway are mutated via error-prone PCR to generate mutant libraries and are flanked by the arms homologous to their neighboring promoters and terminators. These expression cassettes are then assembled using the DNA assembler method.