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Title: Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

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 phylumBacteroidetes. IMPORTANCEMolecular 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 involvesmore » 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.« less
ORCiD logo [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Div.
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth and Environmental Sciences Area
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Div.; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering
Publication Date:
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Accepted Manuscript
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Additional Journal Information:
Journal Volume: 3; Journal Issue: 1; Journal ID: ISSN 2379-5077
American Society for Microbiology
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; genomics; transposons
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