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A Novel Platform for High-Throughput Gene Synthesis to Maximize Recombinant Expression in Escherichia coli

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PCR

Abstract

Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts. The platform is based on a simple gene synthesis method that uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides. This technology incorporates an accurate, automated and cost-effective ligase-independent cloning step to directly integrate the synthetic genes into an effective E. coli expression vector. High-throughput production of synthetic genes is of increasing relevance to allow exploring the biological function of the extensive genomic and meta-genomic information currently available from various sources.

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Correspondence to Ana Filipa Sequeira .

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Sequeira, A.F., Brás, J.L.A., Fernandes, V.O., Guerreiro, C.I.P.D., Vincentelli, R., Fontes, C.M.G.A. (2017). A Novel Platform for High-Throughput Gene Synthesis to Maximize Recombinant Expression in Escherichia coli . In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7060-5_7

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-7059-9

  • Online ISBN: 978-1-4939-7060-5

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