E-Book Overview
A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described.
E-Book Content
Methods in Molecular Biology TM VOLUME 235 E. coli Plasmid Vectors Methods and Applications Edited by Nicola Casali Andrew Preston Plasmid Organization 1 1 The Function and Organization of Plasmids Finbarr Hayes 1. Introduction In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists in an extrachromosomal state for at least part of its replication cycle (1). As this description included bacterial viruses, the definition of what constitutes a plasmid was subsequently refined to describe exclusively or predominantly extrachromosomal genetic elements that replicate autonomously. Plasmids are now known to be present in most species of Eubacteria that have been examined, as well as in Archaea and lower Eukarya (2). Although most of the genetic material that directs the structure and function of a bacterial cell is contained within the chromosome, plasmids contribute significantly to bacterial genetic diversity and plasticity by encoding functions that might not be specified by the chromosome (3) (see Subheading 3). For example, antibiotic resistance genes are often plasmid-encoded, which allows the bacterium to persi