ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

Abstract

The recently evolved field of synthetic biology has revolutionized the way we think of biology as an “engineerable” discipline. The newly sprouted branch is constantly in need of simple, cost-effective and automatable DNA-assembly methods. We have developed a reliable DNA-assembly system, ZeBRα (Zero-Background Redα), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBRα are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBRα combines a “zero-background vector” with a highly efficient in vitro recombination method. The suicide-gene in the vector acts as placeholder, and is replaced by the fragments-of-interest, ensuring the exclusive survival of the successful recombinants. Thereby the background from uncut or re-ligated vector is absent and screening for recombinant colonies is unnecessary. Multiple fragments-of-interest can be assembled into the empty vector by a recombinogenic E. coli -lysate (SLiCE) with a total time requirement of less than 48 h. We have significantly simplified the preparation of the high recombination-competent E. coli -lysate compared to the original protocol. ZeBRα is the least labor intensive among comparable state-of-the-art assembly/cloning methods without a trade-off in efficiency.

Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

“…In all cases, each point represents the average value of multiple experiments. Shown are the data reported here for RecET Gam Δ xonA (filled circles), and published results for RecET Gam 15, 29 (open circles), iVEC 21 (open triangles), ZeBrα 28 (plus signs), Gibson+RecET Gam 30 (open squares), Gibson 30 (stars), and ExoCET 30 (filled diamonds).…”

“…However, as the number of fragments to be assembled increases, cloning efficiency rapidly decreases. 20, 21, 27, 28 In an attempt to compare recombination efficiencies for various numbers of fragments, we have compiled a table (Supporting Information Table S1) of previously reported numbers of recombinants obtained by several laboratories and have compared that data with our results using the RecET system in the Δ xonA background. Although this comparison does have limitations since the methods used in the various laboratories were not uniform, the results are informative and are presented in graphical form in Figure 6.…”

Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters

“…Although these enzyme-free cloning tools provide a number of advantages over other cloning strategies, they still have limitations. For example, these methods usually require a number of specially designed primers, and the assembly capability as well as fidelity drop sharply with increasing fragment size ( Tillett and Neilan, 1999 ; Yuan et al, 2016 ; Liang et al, 2017 ; Richter et al, 2019 ).…”

From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus

“…Extracts from other recA- cloning strains of E. coli have been reported to permit cloning [ 15 ]. Purification of the SLiCE treated cloned DNA prior to transformation has been shown to improve transformation efficiency [ 16 ]].…”