# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 2 Materials and Methods
### High-Cell-Density Cultivation on a 2.5 L Scale  
High-cell-density cultivations of _E. coli_ JM109 with helper plasmid pfhp30 were performed on different scales, including two pre-culture stages. On a 2.5 L scale, the first pre-culture stage was prepared from a cryo-stock in 0.5 L baffled shaking flasks (DURAN\({}^{\circledR}\) Baffled Flask, DWK Life Sciences GmbH, Wertheim, Germany) with 50 mL mineral medium (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, 5 g L\({}^{-1}\) glucose, 1.2 g L\({}^{-1}\) MgSO\({}_{4}\bullet\) 7 H\({}_{2}\)O, trace elements (see Supplementary Materials), 1 mM thiamine, pH 6.7) [31] with 50 mg L\({}^{-1}\) kanamycin as selection marker overnight without pH control at 37 \({}^{\circ}\)C and 150 rpm in a shaking incubator (WIS-20 shaking incubator, Witeg Labortechnik GmbH, Wertheim, Germany). The _E. coli_ JM109 was used in the study of the _E. GmbH, Wertheim, Germany). For the second pre-culture stage, 1 L baffled shaking flasks (DURAN(r) Baffled Flask, DWK Life Sciences GmbH, Wertheim, Germany) with 100 mL mineral medium [31] (5 g L\({}^{-1}\) glucose) with 50 mg L\({}^{-1}\) kanamycin were inoculated with 10 mL of the first pre-culture each and incubated for 6-7 h without pH control at 37 \({}^{\circ}\)C and 150 rpm in a shaking incubator (WIS-20 shaking incubator, Witeg Labortechnik GmbH, Wertheim, Germany). Fed-batch cultivations of _E. coli_ were carried out in a stirred tank bioreactor with a working volume of 2.5 L (KLF Advanced System 3.6 L, Bioengineering AG, Wald, Switzerland). For temperature control, a heating (800 W) and cooling rod and temperature sensor (pt100) were inserted via the base plate. The bioreactor had four equidistant baffles with a passing tube attached, which introduced compressed air into the liquid at 2 vvm through a gassing ring located below the agitator. The agitator shaft was sterile-sealed via a mechanical seal and had two 6-blade disk agitators attached to it. The shaft was driven by a continuously variable electric motor (250 W). Via the lid, other internals, such as pH, a dissolved oxygen probe, and manometer, were introduced to the reactor. Process variables like pH, and dissolved oxygen were controlled via BioSCADALab software (version 6.8, Bioengineering, Wald, 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, pH 6.7) solved in 1.48 L H\({}_{2}\)O 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 25 g L\({}^{-1}\) glucose, 1.2 g L\({}^{-1}\) MgSO\({}_{4}\)\(\bullet\) 7 H\({}_{2}\)O, trace elements, and the sterile-filtered heat-sensitive components (1 mM thiamine, 50 mg L\({}^{-1}\) kanamycin) to 1.6 L [31]. The pH was controlled during fermentation to pH 6.7 by automated titration with 25% NH\({}_{4}\)OH, and the temperature during the batch phase was set to 37 \({}^{\circ}\)C. The stirred tank bioreactor was inoculated with 10-fold concentrated _E. coli_ cells from the second pre-culture stage, aiming for an initial cell concentration of 0.1 g L\({}^{-1}\). The _E. coli_ cells were infected with phagemid particles immediately before transfer into the bioreactor or one hour after the batch phase started, with a multiplicity of infection (MOI) between 10\({}^{-6}\) and 10\({}^{-2}\) ftu cfu\({}^{-1}\) (transforming unit per colony-forming unit). The stirrer speed was adjusted automatically in the batch phase (500 min\({}^{-1}\)-1000 min\({}^{-1}\)) to keep the dissolved oxygen level above 30% air saturation.