Preparation Of Competent Cells Research Articles

Transformation of Xanthomonas axonopodis pv. citri by electroporation

This study describes the use of electroporation for transforming Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus (Citrus spp.) canker. It also evaluates the methodology used for this species under different electrical parameters. The bacterium used in the study (Xac 306) was the same strain used for recent complete sequencing of the organism. The use of a plasmid (pUFR047, gentamycin r) is reported here to be able to replicate in cells of Xac. Following the preparation and resuspension of competent cells of Xac at a density of ~4 x 10(10) cfu/ml, in 10% glycerol, and the addition of the replicative plasmid, an electrical pulse was applied to each treatment. Selection of transformants showed a high efficiency of transformation (1.1 x 10(6) transformants/mug DNA), which indicates an effective, and inverse, combination between electrical resistance (50 W) and capacitance (50 µF) for this species, with an electrical field strength of 12.5 kV.cm-1 and 2.7-ms pulse duration. Besides the description of a method for electroporation of Xac 306, this study provides additional information for the use of the technique on studies for production of mutants of this species.

An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains

This paper describes an efficient bacterial transformation system that was established for the preparation of competent cells, plasmid preparation, and for the storage in bacterial stocks in our laboratory. Using this method, a number of different plasmids have been amplified for further experiments. Competent cells for bacterial transformation were prepared by the calcium chloride method with an optimum concentration of 75 mM. Three different strains of Escherichia coli that were tested are DH5α, TG1 and XL1 blue, and the most efficient strain being XL1 blue. The optimal optical density (OD 600 ) range for competent cell preparation varied for each of the strains investigated, and for XL1 blue it was 0.15-0.45; for TG1 it was 0.2-0.5; and for DH5α it was 0.145-0.45. The storage time of competent cells and its correlation to transformation efficiency has been studied, and the result showed that competent cells can be stored at -20oC for 7 days and at -70oC for 15 days. Three critical alterations to previous methods have been made, which are the changing of the normal CaCl 2 solution to TB solution, the changing of the medium from LB to S.O.C., and addition of DMSO or PEG 8000 during transformation of competent cells with plasmids. Changing the medium from LB to S.O.C., resulted in much faster growth of transformants, and the transformation efficiency was increased. Addition of DMSO or PEG 8000 raised transformation efficiencies by 100-300 fold. Our improved bacterial transformation system can raise the transformation efficiency about 10 3 times, making it becoming a highly efficient bacterial transformation system.

An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains

An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains

Condensed Protocol for Competent Cell Preparation and Transformation of the Methylotrophic Yeast Pichia Pastoris

The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris—spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection. The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heat-shock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably. We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this “hybrid” method are then electroCondensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris

Construction and identification of polycistron adenoviral expression vector PCA13/FasL–IRES–iNOS

To construct the Fas ligand (FasL) and inducible nitric oxide synthase (iNOS) coexpressed PCA13 plasmid used for packing of adenovirus, so as to observe the immunoprotective effect of FasL on the adenovirus vector. By way of the internal ribosome entry site (IRES), gene engineering techniques such as preparation and transformation of competent cells, plasmid extraction, agarose gel electrophoresis and restriction enzymolysis were used for the construction of the polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS, which could coexpress FasL and the iNOS gene after multisubcloning steps. FasL and iNOS connected with the IRES were successfully cloned to the PCA13 plasmid and verified by enzymolysis (600 bp FasL, 1000 bp IRES and 4000 bp iNOS) and the gene sequence was concordant with the gene bank. The polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS was successfully constructed.

Construction and identification of the eukaryotic expression vector of vaccinia virus expressing human interleukin‐2

OBJECTIVE: Cancer gene therapy using interleukin‐2 (IL‐2) has generated much interest because of the potent antitumor effect of this cytokine. There is ongoing research into one promising new gene therapy approach for cancer using the vaccinia virus vector. The purpose of this study was to construct a vaccinia eukaryotic expression vector, pMJ601, which contains human IL‐2 (hIL‐2; pMJ601hIL‐2) and can be used for the treatment of gastric carcinomas. METHODS: Genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, restriction analysis, ligation, preparation of competent cells, transformation and DNA sequence analysis were used to clone the hIL‐2 gene into pBluescript II SK+/– and pMJ601 and identify these vectors. RESULTS: Fragments of hIL‐2 DNA from pLXSN from an EcoR1–BamHI restriction enzyme digest were successfully cloned into pBluescript II SK+/–. In addition, hIL‐2 DNA from pBluescript II SK+/– with a SalI‐BamHI restriction enzyme digest was also successfully cloned into pMJ601. CONCLUSION: Constructing a pMJ601 vector that expresses the hIL‐2 gene is an important step toward being able to treat gastric carcinoma using gene therapy.

Introduction of Plasmid DNA into Cells

This appendix presents a procedure for transformation using calcium chloride, and also an alternate procedure for one-step preparation and transformation of competent cells.

Cryopreservation of competent intact yeast cells for efficient electroporation

We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long-term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non-permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10 degrees C/min and a rapid thawing rate of 200 degrees C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non-electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at -80 degrees C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long-term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation.

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.

Introduction of Plasmid DNA into Cells

This appendix presents a procedure for transformation using calcium chloride, and also an alternate procedure for one-step preparation and transformation of competent cells.

Preparation of competent cells for high-efficiency plasmid transformation of Escherichia coli.

Preparation of competent cells for high-efficiency plasmid transformation of Escherichia coli.

One-step transformation of yeast in stationary phase

A fast yeast-transformation technique has been developed by adding thio compounds to alkali-ion based protocols and incubating at 45 degrees C. This procedure is especially recommended for cells from stationary phase at a density up to 2.5 x 10(8) cells/ml. It involves only one step for the preparation and transformation of competent cells within 30 min. The yield was more than 10(4) transformants/microgram plasmid DNA. This protocol is easy to scale up for many DNA samples and is also applicable for yeast cells from different types of storages.

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

A rapid and convenient method for the preparation and storage of competent bacterial cells.

Journal Article A rapid and convenient method for the preparation and storage of competent bacterial cells Get access C.T. Chung, C.T. Chung Laboratory of Infectious Diseases, National Institute of Allergy and Infectious DiseasesBethesda, MD 20892, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Roger H. Miller Roger H. Miller Laboratory of Infectious Diseases, National Institute of Allergy and Infectious DiseasesBethesda, MD 20892, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 16, Issue 8, 25 April 1988, Page 3580, https://doi.org/10.1093/nar/16.8.3580 Published: 25 April 1988 Article history Received: 15 March 1988 Published: 25 April 1988

Transformation by plasmid and chromosomal DNAs in [formula omitted

An efficient procedure for transformation of H. parainfluenzae by plasmid DNA is described. The procedure involved a new technique for preparation of competent cells that increased the transforming efficiency of a chromosomal str r marker about 20 fold and that of the plasmid amp r about 100 fold. The addition of either Mg 2+ or Ca 2+ promoted the stimulation of plasmid amp r by another factor of 50, however, the chromosomal str r marker was not further affected. The total stimulation of plasmid transformation was a factor of 5000 and the final ratio of amp r to str r activity was 1 to 100. These results suggested that plasmid and chromosomal DNAs have different specific requirements for transformation.