Conditions for efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmid cloning vectors were identified. Three streptomycete plasmid origins of replication function in A. orientalis, as do the apramycin resistance gene from Escherichia coli, the thiostrepton resistance gene from Streptomyces azureus, and the tyrosinase gene from Streptomyces...

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was...

DMSO Inoue transformation buffer (please see Step 1) Chilled to 0°C before use. Plasmid DNA (recombinant plasmid) Construct using one of the methods described in Directional Cloning into Plasmid Vectors, Attaching Adaptors to Protruding Termini, Blunt-ended Cloning into Plasmid Vectors, Dephosphorylation of Plasmid DNA, Addition of Synthetic Linkers to Blunt-ended DNAand Ligating Plasmid and Ta...

BACKGROUND Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB ...

The presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in Moniliophthora perniciosa. The influence of the vector shape (linear or circular), the patterns of plasmid integration in genomic sites and the influence of the promoter used to express the gene marker were also analyzed. The addition of BamHI or NotI increased the number of transforma...

We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombinant plasmid pLC1-hph, which contains the L. edodes transcriptional signals and an Escherichia coli hygromycin B phosphotransferase gene. Protoplasts of L. edodes were treated by the PEG transformation mixture containing 50 units of SalI, which clea...

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA a...

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-d...

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high f...

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation prot...