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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2018-02-01
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| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSystems.00143-17 |
| _version_ | 1818691830579986432 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-17T12:48:08Z |
| format | Article |
| id | doaj.art-7bfe5283e96d4fcdb721b110a8e98eae |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-12-17T12:48:08Z |
| publishDate | 2018-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-7bfe5283e96d4fcdb721b110a8e98eae2022-12-21T21:47:40ZengAmerican Society for MicrobiologymSystems2379-50772018-02-013110.1128/mSystems.00143-17Magic Pools: Parallel Assessment of Transposon Delivery Vectors in BacteriaHualan Liu0Morgan N. Price1Robert Jordan Waters2Jayashree Ray3Hans K. Carlson4Jacob S. Lamson5Romy Chakraborty6Adam P. Arkin7Adam M. Deutschbauer8Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEarth and Environmental Sciences Area, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mSystems.00143-17genomicstransposons |
| spellingShingle | 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 mSystems genomics transposons |
| title | 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_short | Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria |
| title_sort | magic pools parallel assessment of transposon delivery vectors in bacteria |
| topic | genomics transposons |
| url | https://journals.asm.org/doi/10.1128/mSystems.00143-17 |
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