Establishment of uncharacterized plasmids in Escherichia coli by in vitro transposition

Abstract We present a simple approach that permits any circular plasmid, such as uncharacterized plasmids from diverse prokaryotes, to be established in Escherichia coli, thereby facilitating subsequent structural and functional studies. An in vitro transposition reaction is used to introduce a well...

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Bibliographic Details
Published in:FEMS microbiology letters 2002-12, Vol.217 (2), p.249-254
Main Authors: Agron, Peter G., Sobecky, Patricia, Andersen, Gary L.
Format: Article
Language:English
Subjects:
Bacteria
Base Sequence
Biological and medical sciences
Biotechnology
DNA Transposable Elements
E coli
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Genetic Vectors
In vitro transposition
Methods. Procedures. Technologies
Microbiology
Models, Genetic
pGT5
Plasmid isolation
Plasmid recovery
Plasmid rescue
Plasmids
Plasmids - genetics
Prokaryotes
Pyrococcus abyssi
Sequences
Structure-function relationships
Transformation, Bacterial
Transposition
Vectors (cloning, transfer, expression). Insertion sequences and transposons
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Summary:Abstract We present a simple approach that permits any circular plasmid, such as uncharacterized plasmids from diverse prokaryotes, to be established in Escherichia coli, thereby facilitating subsequent structural and functional studies. An in vitro transposition reaction is used to introduce a well-characterized replicon and selectable marker into purified plasmids, which are then used to transform E. coli. The approach was demonstrated using a small 3.4-kb archaeal plasmid and a large 60-kb uncharacterized plasmid from a Gram-negative bacterium. Replicon function in E. coli was tested for each plasmid, and direct sequencing of the large plasmid revealed similarity to restriction–modification systems.
ISSN:0378-1097
1574-6968
DOI:10.1111/j.1574-6968.2002.tb11483.x