# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 2 Materials and Methods
### High-Cell-Density Cultivation on a 1000 L Scale  
The first pre-culture stage was prepared from cryo-stocks in eight 2 L shaking flasks with 250 mL mineral medium [31] (5 g L\({}^{-1}\) glucose) with 50 mg L\({}^{-1}\) kanamycin in each flask and incubated for 12-13 h at 37 \({}^{\circ}\)C and 250 rpm in a shaking incubator. The second pre-culture stage was performed in a 75 L stainless-steel stirred tank bioreactor (Bioengineering AG, Wald, Switzerland) with a working volume of 30 L. The reactor had four baffles and two 6-blade Rushton turbines, a ring sparger, and sensors for relevant process variables like pH, dissolved oxygen, and pressure were controlled via BioSCADALab software (version 6.7 Bioengineering, Wald, Switzerland). The temperature of the stirred tank reactor was controlled by a double jacket for heat transfer. 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 glucose (5 g L\({}^{-1}\)), 1.2 g L\({}^{-1}\) MgSO\({}_{4}\)\(\bullet\) 7 H\({}_{2}\)O, and the sterile-filtered heat-sensitive components. The reactor was inoculated with 2 L of the first pre-culture. The pH was maintained at pH 6.7 throughout the process by automated titration with 25% NH\({}_{4}\)OH, and two six-bladed Rushton impellers obtained a good mixing with a speed of 400 min\({}^{-1}\). The reactor was aerated with compressed air at a supply rate of 0.8 vvm. Cells were infected with phagemid particles at the end of the pre-cultivation with a multiplicity of infection (MOI) of 10\({}^{-3}\) ftu cfu\({}^{-1}\).  
The high-cell-density cultivation was carried out in a 1500 L stainless-steel stirred tank bioreactor (Bioengineering AG, Wald, Switzerland) with a maximum working volume of 1000 L. The reactor had four baffles and two 6-blade Rushton turbines, a ring sparger, and sensors for relevant process variables like pH, dissolved oxygen, and pressure, and was controlled via BioSCADALab software (version 6.7, Bioengineering, Wald, Switzerland). The temperature of the stirred tank reactor was controlled by a double jacket for heat transfer. The reactor was sterilized in situ with 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]. After cooling to 37 \({}^{\circ}\)C, heat-sterilized glucose (20 g L\({}^{-1}\)), 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) were added to achieve a total batch volume of 700 L. A total of 30 L of the second pre-culture was transferred to the main culture, aiming for an initial cell concentration of 0.1 g L\({}^{-1}\). The pH was maintained at pH 6.7 throughout the process by automated titration with 25% NH\({}_{4}\)OH. The dissolved oxygen level was kept above 30% air saturation by adjusting the stirrer speed automatically (161 min\({}^{-1}\)-333 min\({}^{-1}\)), the aeration rate with compressed air (800 NL min\({}^{-1}\)-2000 NL min\({}^{-1}\) corresponding to 0.8-2 vvm), and the pressure (up to 0.9 bar). 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 pumped into the reactor to achieve a growth rate of 0.15 h\({}^{-1}\). The temperature was lowered to 30 \({}^{\circ}\)C at the start of the feeding phase. The process was stopped after 21 h to obtain a better product quality with fewer impurities.