High fidelity one‐pot DNA assembly using orthogonal serine integrases

Abstract

Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. Methods and Major Results:In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. Conclusions and Implications:We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.

Applications of Serine Integrases in Synthetic Biology over the Past Decade

“…Furthermore, orthogonal serine integrase systems enable in vitro assem either linear DNA (Figure 4d) [32] or circular DNA (Figure 4e) [50]. Both can pro large biobricks and assemble several independent modules for the designed applica for example, the biosynthesis of carotenoid [75], erythromycin [76], and the co-expre of chromoproteins [50]. Another serine integrase utilization is in vitro linear DNA assembly for defined purposes.…”

“…Furthermore, orthogonal serine integrase systems enable in vitro assembly of either linear DNA (Figure 4d) [32] or circular DNA (Figure 4e) [50]. Both can produce large biobricks and assemble several independent modules for the designed applications, for example, the biosynthesis of carotenoid [75], erythromycin [76], and the co-expression of chromoproteins [50]. Serine integrase-mediated attP-attB site-specific single integration depends on the pre-existed attB site (pseudosite) [82] on the host chromosome (or post-integrated attB site) and attP site on the exogenous DNA sequence.…”

“…Similarly, Gao et al assembled the erythromycin gene cluster by serine integrases for the production of a secondary metabolite in Streptomyces [76]. Abioye et al created a high fidelity one-pot DNA assembly strategy for the construction of the β-carotenoid pathway [75]. In summary, serine integrase-based linear DNA fragment assembly provides an alternative strategy for molecular cloning.…”

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells

“…Although tyrosine integrases such as Cre/LoxP [ 5 – 8 ] and λ Int [ 5 ] systems have historically been predominantly used, recently, another family of SSRs known as serine integrases has received attention, in great part because of the advantages they present over their counterparts. The main advantages for application in synthetic biology include the shorter length of their attachment sites, unidirectional recombination and nondependence on auxiliary effectors to work [ 9 – 14 ]. Despite the advantages, only a very limited number of functional serine integrases have been available for use in eukaryotic organisms.…”

“…over 30 years ago [ 26 ]. Since then, many works have taken advantage of their recombination capabilities in prokaryotic models [ 15 ], unicellular eukaryotic organisms [ 23 ], and in a few cases, complex multicellular organisms, including mouse and human cells [ 16 , 27 ], and they have even been used as molecular tools for in vitro DNA plasmid assembly [ 14 , 28 ]. More recently, the serine-integrase ability to excise or flip terminators and genes has been more widely applied in the assembly of genetic circuits designed to either modulate gene expression or compute events by permanently rearranging DNA parts based on binary logic [ 15 , 29 – 32 ].…”