Plasmid artificial modification: a novel method for efficient DNA transfer into bacteria. 2011

Tohru Suzuki, and Kazumasa Yasui
The United Graduate School of Agricultural Science, Gifu University, Gifu, Gifu Prefecture, Japan. Suzuki@gifu-u.ac.jp

Bacterial transformation is an essential component of many molecular biological techniques, but bacterial restriction-modification (R-M) systems can preclude the efficient introduction of shuttle vector plasmids into target bacterial cells. Whole-genome DNA sequences have recently been published for a variety of bacteria. Using homology and motif analyses, putative R-M genes can be identified from genome sequences. Introducing DNA methyltransferase genes into Escherichia coli cells causes subsequently transformed plasmids to be modified by these enzymes. We propose a new method, designated Plasmid Artificial Modification (PAM). A PAM plasmid encoding the modification enzymes expressed by the target bacterial host is transformed into E. coli (PAM host). Propagation of a shuttle vector from the PAM host to the target bacterium ensures that the plasmid will be modified such that it is protected from restriction endonuclease digestion in the target bacterium. The result will be a higher transformation efficiency. Here, we describe the use of PAM and electroporation to transform Bifidobacterium adolescentis ATCC15703. By introducing two genes encoding modification enzymes, we improved transformation efficiency 10(5)-fold.

UI MeSH Term Description Entries
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
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
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
D001644 Bifidobacterium A rod-shaped, gram-positive, non-acid-fast, non-spore-forming, non-motile bacterium that is a genus of the family Bifidobacteriaceae, order Bifidobacteriales, class ACTINOBACTERIA. It inhabits the intestines and feces of humans as well as the human vagina.
D014169 Transformation, Bacterial The heritable modification of the properties of a competent bacterium by naked DNA from another source. The uptake of naked DNA is a naturally occuring phenomenon in some bacteria. It is often used as a GENE TRANSFER TECHNIQUE. Bacterial Transformation
D015254 DNA Modification Methylases Enzymes that are part of the restriction-modification systems. They are responsible for producing a species-characteristic methylation pattern, on either adenine or cytosine residues, in a specific short base sequence in the host cell's own DNA. This methylated sequence will occur many times in the host-cell DNA and remain intact for the lifetime of the cell. Any DNA from another species which gains entry into a living cell and lacks the characteristic methylation pattern will be recognized by the restriction endonucleases of similar specificity and destroyed by cleavage. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. DNA Modification Methyltransferases,Modification Methylases,Methylases, DNA Modification,Methylases, Modification,Methyltransferases, DNA Modification,Modification Methylases, DNA,Modification Methyltransferases, DNA
D018274 Electroporation A technique in which electric pulses, in kilovolts per centimeter and of microsecond-to-millisecond duration, cause a loss of the semipermeability of CELL MEMBRANES, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. Depending on the dosage, the formation of openings in the cell membranes caused by the electric pulses may or may not be reversible. Electric Field-Mediated Cell Permeabilization,Irreversible Electroporation,Reversible Electroporation,Electropermeabilisation,Electric Field Mediated Cell Permeabilization,Electroporation, Irreversible,Electroporation, Reversible

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