No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

Abstract

The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes. Alnahhas et al. (Journal of Biological Engineering 2014, 8:28) recently argued that these constraints should be revised because they impose an unnecessary burden on contributors that use homology-based assembly. To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success. Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.

Keywords: Cloning; DNA assembly; Gibson; PCR; Seamless; Synthetic biology; iGEM.

Fig. 1

Overview. a Partially overlapping DNA fragments are transformed into yeast cells together with a linearized plasmid backbone, or fused together by PCR, Seamless or Gibson assembly prior to the transformation. Homologous recombination (HR) enables the fusion of the DNA fragments without transformation. b The assembled plasmid insert contains 4.5 kb DNA encoding two expression units, the TEF1 promoter driving KanR expression, and the TDH3 promoter driving a fusion of the TRP1 and GFP genes. The insert has DNA sequences at the ends that are homologous to the ends of the linearized plasmid backbone (not shown). Assembly success was tested when the insert was broken into two, three, four or five fragments with short or long regions of homology to neighboring fragments or the linearized RS416 plasmid. The transformation used 2 ng or 20 ng of total DNA, including the linearized plasmid. The linearized plasmid DNA was added at the pre-transformation step for Seamless and Gibson assembly

Fig. 2

Charts illustrating the overall success rate and the success rates for each assembly method under different conditions. Success is defined as a 95 % hypergeometric probability or higher that at least one of three clones screened carry a fully functional plasmid. The fraction of failed tests is indicated in grey. The fraction of successful tests is subdivided into different colors to indicate the method used. a All tests. Overall success rate: 65 %. Individual method success rates: 44/56/73/81 % for HR alone, PCR, Seamless and Gibson, respectively. b Tests with 20 ng transformed DNA. Overall: 75 %. Methods: 65/75/83/79 %. c Tests with 20 ng transformed DNA and long regions of overlap between DNA fragments. Overall: 87 %. Methods: 75/83/92/100 %. d Tests performed by A.A. Overall: 69 %. Methods: 61/50/88/75 %. e Tests performed by H.P. Overall: 81 %. Methods: 75/100/62/88 %. f Tests performed by L.T. Overall: 75 %. Methods: 50 %/75 %/100 %/75 %