End Of Logarithmic Growth Phase Research Articles

Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation

The application of safe and cheap inducers is important in the field of fermentation technology, which persuades employing new expression systems. In this study, a Bacillus subtilis expression system was induced by applying starvation and environmental stresses to produce xylanase. The expression plasmid harbors SigB-dependent ohrB promoter. The target gene was expressed by inoculating the recombinant strain into glucose-limited synthetic medium resulting in a sharp increase of xylanase activity at the end of logarithmic growth phase. The recombinant strain was able to express the xylanase enzyme 14-fold higher than that of the control one. The induction was also performed by exposing the recombinant strain to NaCl and ethanol stresses, and heat shock; the strain growing in LB showed 5-, 15- and 6-fold increases in xylanase activity, respectively. The best induction using environmental stresses was achieved by applying the salt stress in the synthetic medium. The maximum expression for NaCl and ethanol stresses occurred after 40 min of induction. All observed inductions were related to activation of SigB protein causing expression of the SigB-dependent xylanase gene. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers.

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.