Abstract
The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 103 cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.
Highlights
Synthetic biology and metabolic engineering require convenient, robust and universal tools to manipulate genetic materials [1,2]
A few of these approaches have reported the assembly of multiple (>3) DNA fragments in a single step. Methods such as the T4 DNA polymerase based sequence and ligation-independent cloning (SLIC) [3], phosphorothioate-based ligase-independent gene cloning (PLICing) [13] and others [16,17,18,19] have only demonstrated the construction of plasmids of less than 8 kb
Other than modifying all the bases in the homologous sequences which increased the cost of primer synthesis, we explored the possibility of decreasing the modification frequency while maintaining a high efficiency of assembly (Figure 2A)
Summary
Synthetic biology and metabolic engineering require convenient, robust and universal tools to manipulate genetic materials [1,2]. The yeast system has been successfully used for the one step assembly of a 19kb fragments into a plasmid or yeast chromosome [15] With these examples, homologous overhang sequences with lengths of 100-500 base pairs were required to increase the assembly efficiency. Homologous overhang sequences with lengths of 100-500 base pairs were required to increase the assembly efficiency This can be a significant challenge where suitable pre-existing sequences in the parental or chemically synthesized templates are required which can restrict the applicability and incur high-cost of synthesis. These approaches are time consuming and labor intensive, are not suited for routine cloning projects
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