Plasmid DNA Preparation and DNA Assembly

Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (QIAGEN) and Econospin columns (Epoch Life Science) according to manufacturer's protocols. PCR reactions were performed with Taq polymerase, PfuUltra Hotstart DNA Polymerase (Agilent Technologies) and Expand High Fidelity PCR System (Roche Life Science) according to manuacturer's protocols. PCR products were purified by Zymoclean Gel DNA Recovery Kit (Zymo Research). All DNA constructs were confirmed through DNA sequencing by Elim Biopharmaceuticals Inc. Restriction enzymes (NEB) and T4 ligase (NEB) were used to digest and ligate the DNA fragments, respectively. BP Clonase II Enzyme Mix, Gateway pDONR221 Vector and LR Clonase II Enzyme Mix (Life Technologies) and the S. cerevisiae Advanced Gateway Destination Vector Kit (Addgene)67 (link) were used to perform Gateway Cloning. Gibson one-pot, isothermal DNA assembly68 (link) was conducted at 10 μl scale by incubating T5 exonuclease (NEB), Phusion polymerase (NEB), Taq ligase (NEB) and 50 ng of each DNA fragment at 50 °C for 1 h to assemble multiple DNA fragments into one circular plasmid. Yeast strains are constructed through homologous recombination and DNA assembly23 (link). Yeast strains and plasmids utilized in this study are listed in Supplementary Data 1 and 2. DNA sequences of genes involved in this work are listed in Supplementary Data 3.

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Li Y, & Smolke C.D. (2016). Engineering biosynthesis of the anticancer alkaloid noscapine in yeast. Nature Communications, 7, 12137.

Publication 2016

QIAprep Spin Miniprep Kit PfuUltra Hotstart DNA Polymerase Expand High Fidelity PCR System Zymoclean Gel DNA Recovery Kit BP Clonase II Enzyme Mix LR Clonase II Enzyme Mix

Biopharmaceuticals Dna constructs Dna polymerase Dna sequences Enzyme Epoch Exonuclease Genes Homologous recombination Ligase Plasmids Restriction enzymes Strains Taq polymerase Vector Yeast

Corresponding Organization :

Other organizations : Stanford University

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Variable analysis

independent variables

Restriction enzymes (NEB)
T4 ligase (NEB)
BP Clonase II Enzyme Mix
Gateway pDONR221 Vector
LR Clonase II Enzyme Mix (Life Technologies)
S. cerevisiae Advanced Gateway Destination Vector Kit (Addgene)
Gibson one-pot, isothermal DNA assembly (incubating T5 exonuclease (NEB), Phusion polymerase (NEB), Taq ligase (NEB) and 50 ng of each DNA fragment at 50 °C for 1 h)

dependent variables

Plasmid DNA prepared using the QIAprep Spin Miniprep Kit (QIAGEN) and Econospin columns (Epoch Life Science)
PCR products purified by Zymoclean Gel DNA Recovery Kit (Zymo Research)
DNA constructs confirmed through DNA sequencing by Elim Biopharmaceuticals Inc.
Yeast strains constructed through homologous recombination and DNA assembly

control variables

Manufacturer's protocols for QIAprep Spin Miniprep Kit, Expand High Fidelity PCR System, Zymoclean Gel DNA Recovery Kit, restriction enzymes, T4 ligase, BP Clonase II Enzyme Mix, LR Clonase II Enzyme Mix, and Gibson one-pot, isothermal DNA assembly

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