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Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

Journal Article · · mSystems
 [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
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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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1434009
Alternate ID(s):
OSTI ID: 1582038
Journal Information:
mSystems, Journal Name: mSystems Journal Issue: 1 Vol. 3; ISSN 2379-5077
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
English

References (35)

Golden Gate Cloning book December 2013
Insights from 20 years of bacterial genome sequencing journal February 2015
Designing Genes for Successful Protein Expression book December 2010
High-throughput bacterial functional genomics in the sequencing era journal October 2015
Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms journal September 2009
Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data journal May 2013
Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries journal November 2011
Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms journal May 2013
Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung journal September 2009
YOABS: yet other aligner of biological sequences—an efficient linearly scaling nucleotide aligner journal March 2012
TIGRFAMs and Genome Properties in 2013 journal November 2012
Brucella abortus strain 2308 putative glucose and galactose transporter gene: cloning and characterization journal May 1997
Pontibacter actiniarum gen. nov., sp. nov., a novel member of the phylum 'Bacteroidetes', and proposal of Reichenbachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Reichenbachia Nedashkovskaya et al. 2003 journal November 2005
Echinicola vietnamensis sp. nov., a member of the phylum Bacteroidetes isolated from seawater journal April 2007
Quantitative phenotyping via deep barcode sequencing journal July 2009
Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants journal October 2009
Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems journal September 2013
Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4 journal November 2016
In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon journal January 2006
Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility journal November 2012
Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. journal January 1982
Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. journal January 1982
Complete nucleotide sequence and transcription of ermF, a macrolide-lincosamide-streptogramin B resistance determinant from Bacteroides fragilis. journal November 1986
Novel Mechanism for Scavenging of Hypochlorite Involving a Periplasmic Methionine-Rich Peptide and Methionine Sulfoxide Reductase journal May 2015
Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons journal May 2015
Target site Selection in Transposition journal June 1997
Transposon-Based Strategies for Microbial Functional Genomics and Proteomics journal December 2003
The RAST Server: Rapid Annotations using Subsystems Technology journal January 2008
Insertion site preference of Mu, Tn5, and Tn7 transposons journal January 2012
Circlator: automated circularization of genome assemblies using long sequencing reads journal December 2015
Accelerated Profile HMM Searches journal October 2011
A One Pot, One Step, Precision Cloning Method with High Throughput Capability journal November 2008
An Integrated Pipeline for de Novo Assembly of Microbial Genomes journal September 2012
Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement journal November 2014
Approaches to querying bacterial genomes with transposon-insertion sequencing journal July 2013

Cited By (3)

Mutant phenotypes for thousands of bacterial genes of unknown function journal May 2018
SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes journal June 2019
Abiotic Factors Promote Cell Penetrating Peptide Permeability in Enterobacteriaceae Models journal November 2019

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Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons
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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
genomics
transposons