Nobuko Nishimura

Cultivation of Escherichia coli Harbouring Hybrid Plasmids

Abstract Cultivation of Escherichia coli transformed with a hybrid plasmid pTREZ1, containing a β-galactosidase (β-gal) gene under the control of the trp promoter, was investigated to develop a control method for effective production of heterologous proteins in bacteria. When glucose in the culture broth was consumed, dissolved oxygen (DO) concentration and respiratory quotient (RQ) rapidly increased. Then the inducer, 3-β-indolylacrylic acid, and casamino acids were added to induce the trp operon. β-Gal production per cell mass reached about 5 U/mg. In addition, RQ rapidly increased when β-gel production ceased, and proved to be a better indicator of E. coli cultivation than DO concentration.

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×108 cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×107 cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×107 cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.