Liu Hualan, Price Morgan N, Waters Robert Jordan, Ray Jayashree, Carlson Hans K, Lamson Jacob S, Chakraborty Romy, Arkin Adam P, Deutschbauer Adam M
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Earth and Environmental Sciences Area, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
mSystems. 2018 Jan 16;3(1). doi: 10.1128/mSystems.00143-17. eCollection 2018 Jan-Feb.
Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term "magic pools." Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum . Molecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a "magic pool." The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA "parts," we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.
转座子诱变与下一代测序相结合（TnSeq）是一种发现细菌基因功能的强大方法。然而，为特定细菌开发合适的TnSeq策略可能既昂贵又耗时。为应对这一挑战，我们描述了一种基于部件的策略，用于构建数百个转座子递送载体文库，我们将其称为“魔法池”。在一个魔法池中，每个转座子载体都有不同的上游序列（启动子和核糖体结合位点）和抗生素抗性标记组合，以及一个随机DNA条形码序列，这使得在诱变实验期间能够追踪每个载体。为了鉴定针对特定细菌的有效载体，我们用一个魔法池对其进行诱变并对产生的插入序列进行测序；然后我们使用这个有效载体来生成一个大型突变文库。我们使用魔法池策略在五个细菌属中构建了转座子突变文库，包括一个门的三个属。分子遗传学对于研究细菌生理学是必不可少的。然而，为任何给定细菌开发功能遗传系统可能很耗时。在这里，我们提出了一种简化的方法，用于为新细菌鉴定有效的转座子诱变系统。我们的策略首先涉及构建数百种不同的转座子载体变体，我们将其称为“魔法池”。使用引入到每个载体设计中的独特DNA条形码并行监测魔法池中每个载体的功效。接下来，我们使用存档的DNA“部件”重新组装一个有效的载体，用于构建全基因组转座子突变文库，该文库适用于使用竞争性生长测定法对基因功能进行大规模研究。在这里，我们展示了魔法池系统在五个细菌属中构建突变文库的实用性。
