Fed-batch culture of Escherichia coli harboring a runaway-replication plasmid.

Abstract

CultureIn fed-batch cultivation of Escherichia coli harboring a runaway-replication plasmid pCP3, runaway replication could not be induced at all by shifting the cultivation temperature from 30 to 42°C, which has normally been used in small-scale experiments. Therefore, cultivation conditions were reexamined by shake-flask cultivations. It was found that the plasmid copy number was restricted to a low level at 25°C but increased gradually at 30°C, and that full runaway replication could be induced at 37°C. Thus, the recombinant E. coli was cultivated at 25°C in fed-batch culture using a minifermenter, and then the temperature was shifted to 37°C. Whenrunaway replication was induced in the middle logarithmic growth phase (2.0 kg dry cell/m3), the gene product was amplified almost 40-fold. Although overproduction of the gene product could be induced in the late logarithmic growth phase (10.4 kg dry cell/m3), the extent of amplification decreased.

Development of new plasmid DNA vaccine vectors with R1-based replicons

“…In particular, we expected greater copy number control before the temperature shift; however, we are not the first group to observe that runaway replication is not completely suppressed at 30°C [14,19]. To further investigate the temperature-dependent behavior of pDMB02-GFP we pursued additional studies at the RNA and protein levels.…”

Control of gene expression from the SUC2 promoter of Saccharomyces cerevisiae with the aid of a glucose analyser

“…In order to solve these problems, we studied the culture conditions of Escherichia coli containing recombinant plasmids such as a runaway replication plasmid (Mizutani et al 1986a(Mizutani et al , 1987a, trp (Kawai et al 1986) and pho promoterbearing ones. Among them, the gene expression from the trp orpho promoter could be derepressed by either tryptophan or phosphate starving in the medium.…”

Mathematical Modelling and Response Characteristics of Runaway Replication For Temperature Shift‐up

“…It has been demonstrated that the gene product encoded by runaway-replication plasmid increases exponentially by induction [2]. Thus, a mathematical model describing both the response of a runaway replication to temperature shift and the cultivation characteristics of the recombinant E. coli is proposed as follows.…”

“…We observed that the gene product increased exponentially in the logarithmic growth phase [2]. For simplicity, we assumed that nutrients, such as carbon source, are well controlled and that their effects on the specific growth rate were negligible.…”

“…Escherichia coli C6OO/pcI857 [2] was transformed by the plasmid pCP3alr and used for the cultivation experiment. Escherichia coli C6OO/pcI857 [2] was transformed by the plasmid pCP3alr and used for the cultivation experiment.…”