Biomaterial Database

Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. 1993

J Quandt, and M F Hynes

Department of Biological Sciences, University of Calgary, Alberta, Canada.

A set of vector plasmids which greatly facilitate gene replacement and reverse genetics in many Gram-negative bacteria was constructed. These vectors are based on the P15A origin of replication (ori) and incorporate sacB from Bacillus subtilis, which is inducible by sucrose and is lethal when expressed in Gram-negative bacteria. The vectors also have a convenient antibiotic-resistance marker (gentamicin resistance) and the lacZ alpha system which allows blue/white selection of cloned fragments. Three different multiple cloning sites, allowing several distinct cloning and gene replacement strategies, are available in the 5' end of lacZ on different vectors. One of these cloning sites, which we synthesised, contains only a NotI-SmaI-NotI sequence; this allows access to most of the restriction sites within the cloned fragment for the purpose of insertion of various cassettes and interposons. The vectors carry the mob region from the broad-host-range plasmid RP4 and are thus mobilizable by conjugation into a wide range of Gram-negative bacteria; since they will not replicate in bacteria other than enterobacteria, they function as 'suicide' vectors. Variants of the vectors carrying the phage lambda cos site were also constructed. We have used these vectors to carry out gene replacement experiments in the fixN region of Rhizobium leguminosarum and have demonstrated that they are extremely useful in eliminating long and tedious screening procedures.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003360	Cosmids	Plasmids containing at least one cos (cohesive-end site) of PHAGE LAMBDA. They are used as cloning vehicles.	Cosmid
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004352	Drug Resistance, Microbial	The ability of microorganisms, especially bacteria, to resist or to become tolerant to chemotherapeutic agents, antimicrobial agents, or antibiotics. This resistance may be acquired through gene mutation or foreign DNA in transmissible plasmids (R FACTORS).	Antibiotic Resistance,Antibiotic Resistance, Microbial,Antimicrobial Resistance, Drug,Antimicrobial Drug Resistance,Antimicrobial Drug Resistances,Antimicrobial Resistances, Drug,Drug Antimicrobial Resistance,Drug Antimicrobial Resistances,Drug Resistances, Microbial,Resistance, Antibiotic,Resistance, Drug Antimicrobial,Resistances, Drug Antimicrobial
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D005804	Genes, Lethal	Genes whose loss of function or gain of function MUTATION leads to the death of the carrier prior to maturity. They may be essential genes (GENES, ESSENTIAL) required for viability, or genes which cause a block of function of an essential gene at a time when the essential gene function is required for viability.	Alleles, Lethal,Allele, Lethal,Gene, Lethal,Lethal Allele,Lethal Alleles,Lethal Gene,Lethal Genes
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D005839	Gentamicins	A complex of closely related aminoglycosides obtained from MICROMONOSPORA purpurea and related species. They are broad-spectrum antibiotics, but may cause ear and kidney damage. They act to inhibit PROTEIN BIOSYNTHESIS.	Gentamicin Sulfate (USP),Gentamycin,G-Myticin,Garamycin,Gentacycol,Gentamicin,Gentamicin Sulfate,Gentamycins,Gentavet,Genticin,G Myticin,GMyticin,Sulfate, Gentamicin