Automating Cloning by Natural Transformation

Jiang, Xinglin, Palazzotto, Emilia, Wybraniec, Ewa, Munro, Lachlan Jake, Zhang, Haibo, Kell, Douglas B ORCID: 0000-0001-5838-7963, Weber, Tilmann and Lee, Sang Yup
(2020) Automating Cloning by Natural Transformation. ACS SYNTHETIC BIOLOGY, 9 (12). pp. 3228-3235.

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Affordable and automated cloning platforms are essential to many synthetic biology studies. However, the traditional <i>E. coli</i>-based cloning is a major bottleneck as it requires heat shock or electroporation implemented in the robotic workflows. To overcome this problem, we explored bacterial natural transformation for automatic DNA cloning and engineering. Recombinant plasmids are efficiently generated from Gibson or overlap extension PCR (OE-PCR) products by simply adding the DNA into <i>Acinetobacter baylyi</i> ADP1 cultures. No DNA purification, competence induction, or special equipment is required. Up to 10,000 colonies were obtained per microgram of DNA, while the number of false positive colonies was low. We cloned and engineered 21 biosynthetic gene clusters (BGCs) of various types, with length from 1.5 to 19 kb and GC content from 35% to 72%. One of them, a nucleoside BGC, showed antibacterial activity. Furthermore, the method was easily transferred to a low-cost benchtop robot with consistent cloning efficiency. Thus, this automatic natural transformation (ANT) cloning provides an easy, robust, and affordable platform for high throughput DNA engineering.

Item Type: Article
Uncontrolled Keywords: automated cloning, benchtop robot, natural transformation, Acinetobacter baylyi ADP1, biosynthetic gene clusters
Depositing User: Symplectic Admin
Date Deposited: 22 Feb 2021 13:22
Last Modified: 18 Jan 2023 22:58
DOI: 10.1021/acssynbio.0c00240
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