Automating Cloning by Natural Transformation

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Affordable and automated cloning platforms are essential to many synthetic biology studies. However, the traditional E. coli-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 Acinetobacter baylyi 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.
Publisher
AMER CHEMICAL SOC
Issue Date
2020-11
Language
English
Article Type
Article
Citation

ACS SYNTHETIC BIOLOGY, v.9, no.12, pp.3228 - 3235

ISSN
2161-5063
DOI
10.1021/acssynbio.0c00240
URI
http://hdl.handle.net/10203/279440
Appears in Collection
CBE-Journal Papers(저널논문)
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