# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 2 Materials and Methods
### High-Cell-Density Cultivation on a 25 L Scale  
For the process transfer to a 25 L scale, the first pre-culture stage was prepared analogous to the 2.5 L scale and incubated for 9 h at 37 \({}^{\circ}\)C and 150 rpm in a shaking incubator (WIS-20 shaking incubator, Witeg Labortechnik GmbH, Wertheim, Germany). The second pre-culture stage was performed in five 1 L baffled shaking flasks (DURAN(r) Baffled Flask, DWK Life Sciences GmbH, Wertheim, Germany) with 200 mL mineral medium [31] (5 g L\({}^{-1}\) glucose) with 50 mg L\({}^{-1}\) kanamycin. The flasks were inoculated with 10 mL of the first pre-culture each and incubated overnight at 37 \({}^{\circ}\)C and 150 rpm in a shaking incubator (WIS-20 shaking incubator, Witeg Labortechnik GmbH, Wertheim, Germany).  
The high-cell-density cultivation was carried out in a 42 L stainless-steel stirred tank bioreactor (Techfors, Infors HT, Bottmingen, Switzerland) with a maximum working volume of 25 L. The bioreactor had four equidistant baffles and three six-bladed Rushton impellers to ensure good mixing and thorough gas dispersion. The reactor was aerated with compressed air at a supply rate of 1 vvm. The temperature of the stirred tank reactor was controlled by a double jacket for heat transfer. Probes for measuring the pH,temperature, and dissolved oxygen concentration were installed at the openings in the lower reactor area. Process variables were regulated by the IRIS Control Software (V5.3, Infors HT, Bottmingen, Switzerland). The heat-resistant media components (13.3 g L\({}^{-1}\) KH\({}_{2}\)PO\({}_{4}\), 4 g L\({}^{-1}\) (NH\({}_{4}\))\({}_{2}\)HPO\({}_{4}\), 1.86 citric acid \(\bullet\) 1 H\({}_{2}\)O, trace elements) [31] were placed into the bioreactor before in situ sterilization (120 \({}^{\circ}\)C, 20 min). After cooling to 37 \({}^{\circ}\)C, the mineral medium was completed with heat-sterilized 20 g L\({}^{-1}\) glucose, 1.2 g L\({}^{-1}\) MgSO\({}_{4}\)\(\bullet\) 7 H\({}_{2}\)O, and the sterile-filtered heat-sensitive components (1 mM thiamine, 50 mg L\({}^{-1}\) kanamycin). For inoculation, _E. coli_ cells from the second pre-culture stage were pumped into the reactor, aiming for an initial cell concentration of 0.1 g L\({}^{-1}\) infected with phagemid particles at a multiplicity of infection (MOI) of 10\({}^{-3}\) ftu cfu\({}^{-1}\). The pH was maintained at pH 6.7 throughout the process by automated titration with 25% NH\({}_{4}\)OH. The stirrer speed was adjusted automatically (300 min\({}^{-1}\)-1000 min\({}^{-1}\)) to keep the dissolved oxygen level above 30% air saturation. Following the initial batch phase, a sterile feeding solution containing 600 g L\({}^{-1}\) glucose, 16 g L\({}^{-1}\) MgSO\({}_{4}\)\(\bullet\) 7 H\({}_{2}\)O, and 14.8 g L\({}^{-1}\) (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) was exponentially added to achieve a growth rate of 0.15 h\({}^{-1}\). When the dissolved oxygen level dropped under 30%, the pressure in the bioreactor was raised up to 1 bar overpressure. The exponential feed profile was stopped, and constant pump rates were set to maintain the dissolved oxygen level above 30%.