A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.     

A rapid and highly efficient method for transformation of sugarcane callus

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.     

A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain.

A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with 'free' mitochondria as judged by marker-enzyme assays and electron microscopy.     

A simple and rapid purification method for Escherichia coli DNA polymerase I.

We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.     

Transformation in Escherichia coli: cryogenic preservation of competent cells.

Escherichia coli cells prepared for transformation by treatment with cold 0.1 M CaCl2 remained viable and competent after storage at -82 degrees C in 15% glycerol; thawed-cell samples yielded up to 10(6) transformants per microgram of plasmid deoxyribonucleic acid.     

A rapid method for preparation of nuclear membranes from mammalian cells.

A simple, rapid procedure for disrupting nuclei of mammalian cells has been characterized. This procedure involves treating isolated nuclear suspensions with the natural polyanion heparin in 0.125 m sucrose buffered between pH 7.0 and 8.0. The rate and extent of nuclear lysis are dependent upon the ratio of nuclei concentration to heparin concentration. This procedure avoids use of intense mechanical disruption, enzymatic digestion, and high salt concentrations for achieving optimum lysis of the nuclei. This method can also be used for large-scale nuclear membrane preparations.     

A simple method for the production of highly competent cells ofAgrobacterium for transformation via electroporation

The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations ofAgrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformedAgrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of threeArgobacterium tumefaciens (EHA101, LBA4404, C58) and twoArgobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin^res, kanamycin^res, and gentamycin^res).     

Escherichia coli recA deletion strains that are highly competent for transformation and for in vivo phage packaging

I describe the construction of a variety of Escherichia coli recA deletion strains designed to facilitate molecular cloning. These recA deletion strains permit the efficient cloning of foreign inserts carried in plasmid, phage, cosmid, phasmid (phage-plasmid hybrid) or phosmid (phage-cosmid hybrid) vectors.     

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.     

A rapid and simple method for extracting intracellular metabolites from Escherichia coli bacteria

The article deals with the development of a new method for the extraction of intracellular glycolytic metabolites from bacterial cells. The study has been made on the culture of E. coli B/r CSH. In accordance with this method, the same bacterial filter is used for both filtration (the removal of the culture fluid) and the extraction of low-molecular components of the cells with perchloric acid. The advantage of this method is the absence of unnecessary operations due to the use of a filter installation designed by the author. Quantitatively, this method yields better and reproducible results. The filtration capacity of different types of filters has been analyzed. The optimal time for the extraction of low-molecular cell components has been determined. A change in the concentration of pyruvate in the process of the cellular cycle of E. coli synchronous culture grown in the presence of glucose has been shown to occur. The newly developed method of extraction can be used not only for E. coli, but also for cells of other types.     

A rapid assay for Escherichia coli 4-thiouridine-tRNA sulfurtransferase.

A simple filter paper assay for the measurement of Escherichia coli 4-thiouridine-tRNA sulfurtransferase activity is described. The assay includes the following procedures: (a) incubation of enzyme with appropriate substrates including unfractionated yeast tRNA and [³⁵S]cysteine, (b) reisolation of tRNA, and (c) binding of tRNA to ion exchange filter papers. The assay can be routinely performed with relatively small sample volumes (0.1 ml) and completed within 14 h. Proof of the validity of the assay is based in part on two experimental observations: (1) tRNA is the predominant ³⁵S-labeled species remaining bound to the filter after extensive washing, and (2) 4-thiouracyl is the predominant thiolated base formed during the assay.     

An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.     

A simple and efficient method for the preparation of GDP-fucose.

A crude extract from hog submaxillary gland was found to synthesize guanosine diphosphate (GDP)-fucose, when incubated with fucose, ATP and GTP, the two enzymatic steps (fucose

fucose-1-phosphate

GDP-fucose) proceeding without noticeable side reactions. Column chromatography on Dowex 1 (HCO₃^?), gel filtration on Sephadex G-15 and preparative paper chromatography gave pure GDP-fucose in an overall yield of 81%. The sugar nucleotide was shown to be active as a glycosyl donor in fucose-incorporating systems.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.     

A rapid method for determining the viability of Escherichia coli cells subjected to the action of low temperatures

When Escherichia coli cells are frozen at a low temperature, various damages appear in them, in particular, in the membranous apparatus. Only stable disorders in the barrier properties of the cytoplasmic membranes are of a critical importance for the cell viability. These disorders should be taken into account when express methods are developed for assaying the viability of bacterial cells. Critical structures (properties) are to be revealed by their analysis after varying the intensity (dose) of the main damaging factors. Provided that the critical feature of each cell has two states (damaged and intact) after the action of extreme factors, its quantitative change correlates in a linear mode with the number of viable cells in the population.