Automating Cloning by Natural Transformation,ACS Synthetic Biology

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Automating Cloning by Natural Transformation
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-11-24 , DOI: 10.1021/acssynbio.0c00240
Xinglin Jiang ₁ , Emilia Palazzotto ₁ , Ewa Wybraniec ₂ , Lachlan Jake Munro ₁ , Haibo Zhang ₃ , Douglas B. Kell _{1,

4} , Tilmann Weber ₁ , Sang Yup Lee _{1,

5}

Affiliation

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.

中文翻译：

通过自然转化自动克隆

负担得起的自动克隆平台对于许多合成生物学研究至关重要。但是，传统的基于大肠杆菌的克隆是一个主要瓶颈，因为它需要在机器人工作流程中实施热冲击或电穿孔。为了克服这个问题，我们探索了细菌自然转化用于DNA自动克隆和工程化的方法。只需将DNA添加到拜氏不动杆菌中即可从Gibson或重叠延伸PCR（OE-PCR）产品有效产生重组质粒ADP1文化。不需要DNA纯化，能力诱导或特殊设备。每微克DNA最多可得到10,000个菌落，而假阳性菌落的数量很少。我们克隆和工程设计了21种各种类型的生物合成基因簇（BGC），长度从1.5到19 kb，GC含量从35％到72％。其中之一是核苷BGC，具有抗菌活性。此外，该方法很容易转移到低成本的台式机器人上，并具有一致的克隆效率。因此，这种自动自然转化（ANT）克隆为高通量DNA工程提供了一个简单，强大且价格合理的平台。

更新日期：2020-12-18

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