Comparison of Multiple Gene Assembly Methods for Metabolic Engineering

摘要

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a Sfz'I-based ligation method. Sfil ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the Sfil method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TALI, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique Sfil restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The Sfil method consists of three steps: first a Sfil linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring Sfil linkers on Sfil digestion; second, the linkers are attached to the desired genes by cloning them into Sfil linker vectors; third, the genes flanked by the three-base linkers, are released by Sfil digestion. In the final step, genes of interest are joined together in a simple one-step ligation.