excess [34, 35]. The effect of glucose and glycerol as carbon source

in combination with lactose as inducer was investigated in diverse

pre-studies [35, 36]. In contrast to glucose, higher lactose uptake

rates could be monitored at higher feeding rates for glycerol feed-

ing. Higher inducer uptake rates are believed to boost heterologous

protein production [31, 37, 38]. Chemostat cultivations on glyc-

erol–lactose systems indicated long-term productivity can be

boosted compared to glucose-lactose co-feeding [36]. Based on

our previous investigations and stated arguments in literature, glyc-

erol and lactose show a suitable combination to enable stable long-

term cultivations. Therefore, we focus the method workflow given

in this protocol to the utilization of glycerol and lactose. In the

following sections, a guideline is given on how to set up a cascaded

continuous cultivation with E. coli BL21(DE3).

2

Materials

2.1

Host Cells

Competent E. coli BL21(DE3) host cells (Life technologies, Carls-

bad, CA, USA) are used. Transformation of target plasmid in E. coli

host cells is described in detail elsewhere [39–41]. Depending on

the plasmid backbone, the antibiotic resistance has to be adapted

for cultivation.

2.2

Required Media

A defined medium referred to DeLisa et al. is used for all cultiva-

tions [42]. Medium composition is summarized in Table 1.

2.3

Required Devices

for Cultivation

1. For preculture cultivation, an incubation shaker is required,

allowing shaking possibilities up to 250 rpm and a temperature

control in the range of 30–40
C (e.g., Multitron shaker,

Infors, Bottmingen, Switzerland).

2. For cascaded continuous cultivations (at least) two continu-

ously operated stirred-tank reactors are required:

(a)

Both reactors require separate control systems.

(b)

Reactors need to be capable of stirring to at least

1400 rpm.

(c)

Reactors need to be capable of supplying pressurized air at

minimum 2 vvm.

(d)

Additional oxygen supply must be present to mix gas flows

to a desired ratio.

(e)

A PI- or PID-controller needs be integrated in the process

control system to keep dO2 > 30%.

(f)

Devices must have a working volume larger than 250 mL;

for example, Minifors 2 reactor systems, Labfors 4 or

5 reactor systems or DASGip cultivation systems could

be used. Smaller devices showed process deviations due to

sample volume taken.

A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3)

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