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Recombination-Based DNA Assembly and Mutagenesis Methods for Metabolic Engineering

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Microbial Metabolic Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 834))

Abstract

In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis protocol enhanced by homologous recombination is also described.

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Authors and Affiliations

  1. Life Technologies Corporation, Carlsbad, CA, USA

    Xiquan Liang, Lansha Peng, Billyana Tsvetanova, Ke Li, Jian-Ping Yang, Tony Ho, Josh Shirley, Liewei Xu, Jason Potter, Wieslaw Kudlicki, Todd Peterson & Federico Katzen

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  1. Xiquan Liang
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  2. Lansha Peng
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  3. Billyana Tsvetanova
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  4. Ke Li
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  5. Jian-Ping Yang
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  6. Tony Ho
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  7. Josh Shirley
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  8. Liewei Xu
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  9. Jason Potter
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  10. Wieslaw Kudlicki
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  12. Federico Katzen
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Correspondence to Federico Katzen .

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Editors and Affiliations

  1. Central Research & Development, E.I. Du Pont de Nemours and Company, Route 141 and Henry Clay Rd., Wilmington, 19880, Delaware, USA

    Qiong Cheng

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Liang, X. et al. (2012). Recombination-Based DNA Assembly and Mutagenesis Methods for Metabolic Engineering. In: Cheng, Q. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 834. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-483-4_8

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  • DOI: https://doi.org/10.1007/978-1-61779-483-4_8

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-482-7

  • Online ISBN: 978-1-61779-483-4

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