Minoru Suga

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30°C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1×109 cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2×107 transformants/µg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non‐permeating cryoprotectant such as sorbitol. Copyright

A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells

A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10–15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 106 transformants/µg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long‐term for more than 3 months without any significant loss of efficiency. Copyright

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°C/min and a rapid thawing rate of 200°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°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. Copyright

Gene transfer and protein release of fission yeast by application of a high voltage electric pulse

A high voltage electric pulse can be applied to induce the uptake of DNA into cells and the release of protein from cells. In transformation procedures, electroporation is widely used since the technique is simple, rapid, reproducible, and highly efficient. In extraction of protein, on the other hand, electroextraction has many advantages over other conventional extractions. We have developed a highly efficient method for the electroporation of fission yeast. In particular, application of a high voltage electric pulse to fission yeast improves the cellular uptake and release of macromolecules controlled by both osmotic conditions and electric field strength.

High osmotic stress improves electro-transformation efficiency of fission yeast

A preincubation of fission yeast cells with hyperosmotic solution improved the electro-transformation efficiency. The efficiency increased approximately five-fold when the cells were preincubated with 2.0 M sorbitol and 1.5 M NaCl at 30 degrees C for 60 min before an applied high electric pulse. Losses in the efficiency of the cells after hyperosmotic stress above 2.5 M sorbitol and 2.0 M NaCl were directly related to the marked reduction of viability. The efficiency at 2.0 M sorbitol gradually increased until 60 min of the preincubation period, but longer exposure resulted in a gradual decrease. On the other hand, when the cells of the osmotic-sensitive mutant were preincubated with isosmotic solution of 0.5 M sorbitol, the efficiency was also dramatically increased by approximately 15-fold. These improvements in efficiency were observed in sublethal conditions of osmotic stress regardless of osmoticums and strains.

Orientation and Characteristic Movement of Yeast Cells Induced by Homogeneous Electric Field

A cylindrical yeast cell ( Schizosaccharomyces pombe ) oriented itself parallel or perpendicular to an alternating homogeneous electric field, depending upon the applied frequency. The turnover frequency was about 65 MHz for the living cells in a non-electrolyte solution but about 1.5 MHz for the dead cells in an electric field. Further, the lower the cell activity due to the refrigeration effect, the lower the turnover frequency was. Similar phenomena were observed for budding yeast cells ( Saccharomyces cerevisiae ). Therefore, these phenomena can be used to investigate the cell activity in the solution. We also found a second turnover frequency of about 15 MHz for the dead cylindrical cells. Moreover, when two living cylindrical cells were placed in contact with each other, they exhibited characteristic movement at applied frequencies near the first turnover frequency.

Non-invasive, electro-orientation-based viability assay using optically transparent electrodes for individual fission yeast cells

A non-invasive assay of cylindrical yeast cell viability based on electro-orientation (EO) in an alternating electric field was developed, in which cell viability can be determined by each cells EO direction without the need for reagents. A cell suspension of a few microliters was sandwiched between a pair of optically transparent indium-tin-oxide (ITO) plate electrodes. Observation under a light microscope enabled easy identification of EO based on cell shape, e.g., cells were standing upright and appeared perfectly circular when oriented parallel to the electric field direction (standing position), and they were lying flat and had an elongated shape when oriented perpendicular to the field (lain-down position). The alternative EO positions of living or dead cells were dependent on the applied frequency: opposite EO positions were obtained by applying an AC voltage of 1.5V at 10MHz; at which point, only living cells rapidly attained a standing position, whereas dead cells were lain-down within 10s. All the cells EO positions agreed well with a viability assay by florescence staining. Therefore, at the single-cell level and fluorescently label-free, it was possible to simply and accurately determine whether individual cells were alive or dead based on their shape.

Myotube cell-based 2D-SPR sensor for insulin and its analog detection

We have previously reported that 2D-SPR sensor can observe the intracellular reactions occurring near the bottom of rat myoblast cells cultured on a gold chip upon human insulin stimulation without probes [1]. In this paper, the myotube cells differentiated from myoblast cells were used as the molecular recognition element of the cell-based 2D-SPR for wild-type insulin and its analog. Though the time-course response pattern of the myotube-based sensor for insulin was similar to that of the myoblast-based sensor, the detection limit for human insulin of the myotube-based sensor increased approximately 50-fold as compared with the myoblast-based sensor. Detection range for insulin was 0.1-0.5 μM. The myotube-based sensor was further applied to insulin analog detection. Though the detection range for the analog was the same as for wild-type insulin, the time-course pattern of SPR signal was clearly different as compared with that for wild-type insulin. This difference of response pattern was considered as the difference of the relative amount of monomeric form of insulin and insulin analog. These results suggested the useful strategy for sensitivity improvement and the high discrimination ability of the cell-based 2D-SPR sensor. Correspondence to: Yuki Shiraishi and Hiroaki Shinohara, Graduate School of Innovative Life Science, University of Toyama, Toyama City930-8555, Japan, Email: ebihadoku@gmail.com and hshinoha@eng.u-toyama.ac.jp