Efficient transformation ofKlebsiella oxytoca by electroporation

A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a high transformation efficiency. The highest efficiency of 1.6 × 10⁶ transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD₆₀₀ of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF.     

Transformation of Kluyveromyces lactis by Electroporation

The physical and biological parameters involved in efficient transformation of Kluyveromyces lactis by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 10⁶ and 10⁷ transformants per μg of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid.     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

Improved conditions for the transformation by electroporation of the extracellular polysaccharide-producing methylotroph Methylobacillus sp.

The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 10³ times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times.     

Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

Improved transformation of Pseudomonas putida KT2440 by electroporation

Summary An electric field-mediated transformation (i.e. electroporation) was performed to determine optimal conditions for P. putida transformation. The effects of culture age, electroporation buffer composition, electric field strength, pulse time constant and DNA concentration on transformation efficiency were examined. When plasmid DNA of 8 to 11 kb in size was used with an electroporation buffer containing 1 mM HEPES (pH 7.0), maximum transformation efficiency of 1.0 × 10⁷ transformants/g DNA was obtained at field strength of 12 kV/cm with pulse time of 2.5 millisecond. A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude. A linear relationship was observed between growth phase and transformation efficiency up to OD₆₀₀ = 2.0. This reliable and simple method should be useful for introduction of plasmid DNA into intact P. putida cells.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10⁴ transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Targeted integrative transformation of Candida tropicalis by electroporation

Summary A method for integrative transformation of the diploid yeast Candida tropicalis by electroporation has been developed. By linearizing the transforming plasmid DNA containing the URA3 gene prior to electroporation of recipient cells, its integration was targeted to a specific locus in the genome, resulting in single or multiple tandem integrations. The optimal electroporation conditions for this yeast were established and include an electric pulse of 2.25 kV/cm for a duration of 50 ms. Using these conditions, Ura⁺ transformants were readily obtained at a high frequency (45 transformants/g DNA) as the result of targeted integration of the URA3 gene containing plasmid DNA at the chromosomal ura3 locus. The number of transformants resulting from this procedure is comparable to that achieved with a recently reported spheroplast transformation procedure for C. tropicalis; in addition, it offers the advantages of being simple, rapid and reproducible.     

Transformation of Intact Aspergillus niger by Electroporation

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.     

An improved protocol for electroporation of Oenococcus oeni ATCC BAA‐1163 using ethanol as immediate membrane fluidizing agent

Aims: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA‐1163 strain. Methods and Results: The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 10³ per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm^?1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions: An effective protocol to transform O. oeni ATCC BAA‐1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA‐1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. Significance and Impact of the Study: This is the first report of a successful electroporation of O. oeni ATCC BAA‐1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.     

Bacterial transformation using micro-shock waves

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 μm thick metal foil, 200 mM CaCl₂, 1 ng/μl plasmid DNA concentration, and 1 × 10⁹ cell density. The highest transformation efficiency achieved (1 × 10⁻⁵ transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1 × 10⁻⁶ transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonellatyphimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation.     

Genetic transformation of Clostridium botulinum hall a by electroporation

Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Em^r) and chloramphenicol (Cm^r) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (10³ transformants/g of DNA) when 1 g of plasmid DNA and 4 × 10⁸ CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

High-efficiency transformation of Bacillus subtilis NB22, an antifungal antibiotic iturin producer,by electroporation

High-voltage electroporation was used to transform Bacillus subtilis NB22, an antifungal antibiotic producer, reaching the efficiency of 10⁷ transformants/μg plasmid DNA. Transformation frequency was dependent on the composition of the electroporation solution, the electrical field strength and the cell concentration. Addition of polyethylene glycol (PEG) and mannitol in the transformation solution was critical for a high efficiency of transformation.     

Transformation of Bacillus brevis 47 by electroporation

Summary Bacillus brevis 47 was successfully and reproducibly transformed with pUB110 plasmid DNA by electroporation with an efficiency of 10⁴ transformants per g of DNA. This represents a 10-fold improvement over the chemical transformation method previously used for this organism.     

Optimized transformation of Streptomyces sp. ATCC 39366 producing leptomycin by electroporation

Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam ^? ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×10² CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.