Automating Cloning by Natural Transformation

Xinglin Jiang, Emilia Palazzotto, Ewa Wybraniec, Lachlan Jake Munro, Haibo Zhang, Douglas Kell, Tilmann Weber*, Sang Yup Lee

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume9
Issue number12
Pages (from-to)3228-3235
Number of pages8
ISSN2161-5063
DOIs
Publication statusPublished - 2020

Keywords

  • Automated cloning
  • Benchtop robot
  • Natural transformation
  • Acinetobacter baylyi ADP1
  • Biosynthetic gene clusters

Fingerprint

Dive into the research topics of 'Automating Cloning by Natural Transformation'. Together they form a unique fingerprint.

Cite this