Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.
TLDR
P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.Abstract:
Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.read more
Citations
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Journal ArticleDOI
Studies on transformation of Escherichia coli with plasmids
Douglas Hanahan,Douglas Hanahan +1 more
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
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A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria
TL;DR: In this paper, a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli was developed, which can utilize any gram negative bacterium as a recipient for conjugative DNA transfer.
Book ChapterDOI
Use of T7 RNA polymerase to direct expression of cloned genes.
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Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
TL;DR: The tight regulation of the PBAD promoter is exploited to study the phenotypes of null mutations of essential genes and the use of pBAD vectors as an expression system is explored.
Journal ArticleDOI
A rapid boiling method for the preparation of bacterial plasmids
David S. Holmes,Michael Quigley +1 more
TL;DR: A simple and rapid method for preparing plasmids for restriction enzyme analysis has been developed and can be readily adapted for the preparation of plasmid from liter cultures with quantitative yields.
References
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Journal ArticleDOI
Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.
Francisco Bolívar,Raymond L. Rodriguez,Patricia J. Greene,Mary C. Betlach,Herbert L. Heyneker,Herbert W. Boyer,Jorge H. Crosa,Stanley Falkow +7 more
TL;DR: In vitro recombination techniques were used to construct a new cloning vehicle, pBR322, which is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc).
Journal ArticleDOI
Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA
TL;DR: Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R.factor DNA in this system.
Journal ArticleDOI
SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM
TL;DR: Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE(1)-protein complex after treatment with these various agents is largely owing to an induced transition of ColE (1) DNA from the supercoiled to the open circular state.
Journal ArticleDOI
Construction of Biologically Functional Bacterial Plasmids In Vitro
TL;DR: Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules.