Cloning of Cytosolic Diterpenoid Biosynthetic Genes

The cloning of cytosolic diterpenoid boost, tHMGR and GGPPS, and cytosolic TS are described in previous study^{28 (link)}. Taxus TS1, TS2, T5αH, TAT, T10βΗ, DBAT, T13αΗ, and TAX19 genes (Supplementary Table 9) were synthesized by Gen9 Bio (San Jose, CA) and amplified by PCR using Q5 High-Fidelity DNA Polymerase (New England BioLabs) and gene-specific primers from Integrated DNA Technologies (Supplementary Table 10). PCR amplicons were ligated with AgeI- and XhoI- (New England BioLabs) linearized pEAQ-HT vector^{27 (link)} using HiFi DNA assembly mix (New England BioLabs). Constructs were transformed into 10-beta competent E. coli cells (New England BioLabs). Plasmid DNA was isolated using the QIAprep Spin Miniprep Kit (Qiagen) and sequence verified by Elim Biopharm.

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Liu J.C., De La Peña R., Tocol C, & Sattely E.S. (2024). Reconstitution of early paclitaxel biosynthetic network. Nature Communications, 15, 1419.

Publication 2024

Q5 High-Fidelity DNA Polymerase HiFi DNA assembly mix 10-beta competent E. coli cells QIAprep Spin Miniprep Kit

Corresponding Organization :

Other organizations : Stanford University

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Variable analysis

independent variables

Cloning of cytosolic diterpenoid boost, tHMGR and GGPPS, and cytosolic TS
Synthesis of Taxus TS1, TS2, T5αH, TAT, T10βΗ, DBAT, T13αΗ, and TAX19 genes

dependent variables

Not explicitly mentioned

control variables

Q5 High-Fidelity DNA Polymerase
Gene-specific primers from Integrated DNA Technologies
AgeI- and XhoI-linearized pEAQ-HT vector
HiFi DNA assembly mix
10-beta competent E. coli cells
QIAprep Spin Miniprep Kit
Sequence verification by Elim Biopharm

controls

Positive control: Not mentioned
Negative control: Not mentioned

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