Transformation of Acetobacter xylinum with plasmid DNA by electroporation.

Genetic analysis of Acetobacter xylinum, a cellulose-synthesizing bacterium, has been limited by lack of a successful transformation method. Transformation of A. xylinum was attempted using two broad-host-range plasmids (pUCD2 and pRK248) and a variety of transformation methods. Methods using CaCl2, freeze/thaw treatments, and polyethylene glycol were unsuccessful. Transformation of a cellulose-negative strain of A. xylinum with plasmid DNA has been achieved with high-voltage electroporation. Electroporation conditions of 25 microF capacitance, 2.5 kV, 400 ohms resistance, and pulse lengths of 6-8 ms were applied to a cell/DNA mixture in a 0.2-cm cuvette. Plasmid pUCD2 transformed at an efficiency of 10(6)-10(7) transformants/micrograms DNA and pRK248 yielded 10(5) transformants/micrograms DNA. The frequency of transformation increased linearly with increasing DNA concentration, while transformation efficiency remained constant. pUCD2 was recovered from transformants following chloramphenicol amplification and observed by agarose gel electrophoresis. Both plasmids could be reisolated from Escherichia coli after back-transformation with alkaline lysis DNA preparations from Acetobacter transformants. Electro-transformation of A. xylinum with plasmid DNA suggests its potential use for analysis of the A. xylinum genome.     

Transformation of bacteria by electroporation

The introduction of DNA into bacteria by transformation is an essential step in the construction of recombinant strains. Recently, electroporation, or electropermeabilization, in which a brief high voltage electric discharge is used to render cells permeable to DNA, has revolutionized the transformation of bacteria. The technique is fast, simple, reproducible, frequently gives very high transformation frequency and appears to be applicable to a wide range of bacterial types previously thought untransformable. The technique can also offer advantages for transformable bacteria such as Escherichia coli.     

Transformation ofHaemophilus influenzae following electroporation with plasmid and chromosomal DNA

Naturally transformable species, such asHaemophilus influenzae, have evolved systems for the efficient uptake and integration of DNA from the surrounding environment. We compared this competence-dependent DNA uptake system to electroporation, which has been widely used in the past few years to introduce DNA into cells, for transformingHaemophilus influenzae. Electroporation improved transformation efficiency when noncompetent cells were used and when DNA lackingHaemophilus-specific uptake sequences was used for transformation of competent cells. An increase in plasmid-to-chromosome recombination was seen when plasmid DNA containing chromosomal inserts was introduced into competent cells by electroporation, as observed previously for natural transformation.     

Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation

In this paper, the influence of various parameters on plasmid transformation by electroporation of Staphylococcus epidermidis Tü3298 was investigated. Cell growth conditions, various concentrations and forms of plasmid DNA, field strength, pulse duration and media for electroporation and regeneration were tested. In order to obtain optimal transformation efficiency, the cells were incubated for 30 min with DNA before pulsing. With the optimized procedure, other staphylococcal species such as S. aureus, S. staphylolyticus and S. carnosus were transformed with an efficiency up to 3 X 10(5) transformants per micrograms pC194 plasmid DNA.     

Transformation of the coryneform bacterium cellulomonas flavigena with plasmid DNA via electroporation

Summary Using electroporation we have transformed Cellulomonas flavigena with a shuttle vector (pJA85) derived from the E. coli plasmid pUC8 and the Brevibacterium lactofermentum plasmid pULRS8. Upon transformation this plasmid was found to be stable, not to undergo detectable deletion, and to express antibiotic resistance markers originating in Brevibacterium.     

Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation

Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.     

Transformation of Mycoplasma hominis by plasmid pAM120 using electroporation

Mycoplasma hominis was transformed by electroporation with plasmid pAM120 containing the transposon Tn916 that carried the tetM gene responsible for the resistance to tetracycline. The frequency of transformation was 10(-7)-10(-8) colony-forming units (CFU) per 10 micrograms of plasmid DNA. The PCR analysis of transformed DNA confirmed the transposon integration into the mycoplasma genome.     

Transformation of Escherichia coli with large DNA molecules by electroporation.

We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e. molecules > 100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA. Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

Transformation of Bacillus licheniformis by plasmid DNA

A system for obtaining regenerating protoplasts of highly active Bacillus licheniformis 1001 strain was developed. Transformation of protoplasts by pUB110 and pminiKC plasmids (constructed from plasmids pUB110 and pC194) leading to the expression of kanamycine resistance, was demonstrated. It is supposed that in Bac licheniformis, the pminiKC plasmid is integrated into cellular chromosome, in contrast to pUB110 and parental Bac subtilis (pminiKC) strain. Still, the integrated plasmid seems to be not completely under control of the host chromosome. As a result of such integration, the plasmid conversion takes place, resulting in alteration of cytokinesis (filament formation) and sporulation, but not interfering with the ability to produce antibiotic bacitracin.     

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.     

Transformation of Dictyostelium discoideum with plasmid DNA

DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.     

Transformation of Bacillus polymyxa with plasmid DNA

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Transformation of Kluyveromyces lactis by killer plasmid DNA

A single plasmid of 55 kilobases was found in crude cell lysates of each of nine strains of Rhodospirillum rubrum. Restriction endonuclease analysis showed identical fragment patterns with a given nuclease for all plasmids except one, for which an additional EcoRI site was observed. Elimination of the plasmid required that the cells be passaged several times in 25 mM calcium-containing medium, followed by at least two passages under photosynthetic growth conditions in low-calcium medium before treatment with ethyl methanesulfonate. The resulting plasmidless mutants only grew aerobically and were all incapable of pigment formation and photosynthetic growth, suggesting that plasmid DNA is required for photosynthetic competence in R. rubrum.     

Transformation of pathogenic pseudomonas by plasmid DNA]

The possibility has been shown of the genetical transformation of Pseudomonas mallei strains by the purified DNA of the plasmids RSF1010, pES154, pBS222 and pBR325. The frequency of transformation varied from 1.2 x 10(1) to 2.0 x 10(2) depending on the plasmid DNA and transformation technique used in the experiments. Pseudomonas pseudomallei cells could not be transformed by the methods described in the paper.