Abstract
Gibson one-step, isothermal assembly method (Gibson assembly) can be used to efficiently assemble large DNA molecules by in vitro recombination involving a 5′-exonuclease, a DNA polymerase and a DNA ligase. In the past few years, this robust DNA assembly method has been widely applied to seamlessly construct genes, genetic pathways and even entire genomes. Here, we expand this method to clone large DNA fragments with high GC contents, such as antibiotic biosynthetic gene clusters from Streptomyces . Due to the low isothermal condition (50 °C) in the Gibson reaction system, the complementary overlaps with high GC contents are proposed to easily form mismatched linker pairings, which leads to low assembly efficiencies mainly due to vector self-ligation. So, we modified this classic method by the following two steps. First, a pair of universal terminal single-stranded DNA overhangs with high AT contents are added to the ends of the BAC vector. Second, two restriction enzyme sites are introduced into the respective sides of the designed overlaps to achieve the hierarchical assembly of large DNA molecules. The optimized Gibson assembly method facilitates fast acquisition of large DNA fragments with high GC contents from Streptomyces.
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References
Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70(3):461–477
Bode HB, Muller R (2005) The impact of bacterial genomics on natural product research. Angew Chem Int Edit 44(42):6828–6846
Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–347
Gibson DG, Benders GA, Andrews-Pfannkoch C et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220
Li L, Zhao YW, Ruan LJ et al (2015) A stepwise increase in pristinamycin II biosynthesis by Streptomyces pristinaespiralis through combinatorial metabolic engineering. Metab Eng 29:12–25
Casini A, MacDonald JT, De Jonghe J et al (2014) One-pot DNA construction for synthetic biology: the modular overlap-directed assembly with linkers (MODAL) strategy. Nucleic Acids Res 42:e7
Kieser T, Bibb MJ, Butter MJ et al (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich
Acknowledgement
This work was financed by the grants from National Natural Science Foundation of China (31630003, 31421061, 31370081, and 31570072) and the National Basic Research Program of China (2012CB721103).
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Li, L., Jiang, W., Lu, Y. (2018). A Modified Gibson Assembly Method for Cloning Large DNA Fragments with High GC Contents. In: Jensen, M.K., Keasling, J.D. (eds) Synthetic Metabolic Pathways. Methods in Molecular Biology, vol 1671. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7295-1_13
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DOI: https://doi.org/10.1007/978-1-4939-7295-1_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7294-4
Online ISBN: 978-1-4939-7295-1
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