Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

ABSTRACT 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 p...

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Autores principales:	Hualan Liu, Morgan N. Price, Robert Jordan Waters, Jayashree Ray, Hans K. Carlson, Jacob S. Lamson, Romy Chakraborty, Adam P. Arkin, Adam M. Deutschbauer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	genomics transposons Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7bfe5283e96d4fcdb721b110a8e98eae
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spelling	oai:doaj.org-article:7bfe5283e96d4fcdb721b110a8e98eae2021-12-02T18:15:43ZMagic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria10.1128/mSystems.00143-172379-5077https://doaj.org/article/7bfe5283e96d4fcdb721b110a8e98eae2018-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00143-17https://doaj.org/toc/2379-5077ABSTRACT 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 Bacteroidetes. IMPORTANCE 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.Hualan LiuMorgan N. PriceRobert Jordan WatersJayashree RayHans K. CarlsonJacob S. LamsonRomy ChakrabortyAdam P. ArkinAdam M. DeutschbauerAmerican Society for MicrobiologyarticlegenomicstransposonsMicrobiologyQR1-502ENmSystems, Vol 3, Iss 1 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	genomics transposons Microbiology QR1-502
spellingShingle	genomics transposons Microbiology QR1-502 Hualan Liu Morgan N. Price Robert Jordan Waters Jayashree Ray Hans K. Carlson Jacob S. Lamson Romy Chakraborty Adam P. Arkin Adam M. Deutschbauer Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
description	ABSTRACT 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 Bacteroidetes. IMPORTANCE 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.
format	article
author	Hualan Liu Morgan N. Price Robert Jordan Waters Jayashree Ray Hans K. Carlson Jacob S. Lamson Romy Chakraborty Adam P. Arkin Adam M. Deutschbauer
author_facet	Hualan Liu Morgan N. Price Robert Jordan Waters Jayashree Ray Hans K. Carlson Jacob S. Lamson Romy Chakraborty Adam P. Arkin Adam M. Deutschbauer
author_sort	Hualan Liu
title	Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
title_short	Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
title_full	Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
title_fullStr	Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
title_full_unstemmed	Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria
title_sort	magic pools: parallel assessment of transposon delivery vectors in bacteria
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/7bfe5283e96d4fcdb721b110a8e98eae
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Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

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