Establishment of uncharacterized plasmids in Escherichia coli by in vitro transposition.
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.
Agron
Peter G
PG
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, L-441, 7000 East Avenue, Livermore, CA 94550, USA.
Sobecky
Patricia
P
Andersen
Gary L
GL
D001483:Base Sequence; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements