# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 3 Results
### Transfer to a 1000 L Scale  
We further studied the production of CSF1_6106 ssDNA in a high-cell-density cultivation of _E. coli_ JM109 pfhp30 in a 1000 L stirred tank bioreactor. First, cells were grown in the first pre-culture stage in shaking flasks. The second pre-culture stage was performed as a batch process in a stirred tank bioreactor on a 30 L scale. Glucose was consumed within 10 h. Cells were infected with CSF1_6106 phagemid particles after 13 h and transferred to the 1000 L reactor. The fed-batch process data, including cell dry weight concentration, temperature, feeding profile, and ssDNA concentration, isolated from samples and applied to agarose gel electrophoresis are shown in Figure 4. The total process run time was reduced to 21 h. As shown before, impurities were observed after 21 h when the _E. coli_ cells moved to the stationary phase. The reduced process time should lead to fewer DNA impurities and facilitate product purification. Figure 3: High-cell-density cultivation of _Escherichia coli_ JM109 pfhp30 in a 25 L stirred tank bioreactor at 37 \({}^{\circ}\)C with temperature shift to 30 \({}^{\circ}\)C at the beginning of the feeding (indicated by the vertical black line). Simultaneously with inoculation, cells were infected with CSF1_4843 phagemid particles (black circles) or with CSF1_6106 phagemid particles (blue diamonds) with MOI = 10\({}^{-3}\) ftu cfu\({}^{-1}\). Error bars refer to standard deviations of triplicates in measurements. **(a)** Cell dry weight concentration, temperature, and feed profile. **(b)** ssDNA concentration was calculated from the measured absorbance at 260 nm. Then, samples were analyzed using agarose gel electrophoresis to estimate the fraction of actual product of ssDNA using ImageLab (gray circles: CSF1_4843; gray diamonds: CSF1_6106). **(c)** AGEP of the ssDNA (scaffold) produced in this cultivation. **(d)** Class average micrographs of the folded 42-helix bundles. The initial glucose of 20 g L\({}^{-1}\) was consumed after 8.9 h, and the exponential feeding phase started. A maximal cell dry weight concentration of 48 g L\({}^{-1}\) was obtained within 21 h. From the cell dry weight concentration curve, it is visible that the cells were still in an exponential growth phase and did not become stationary before harvest. The feeding profile was exponential during the whole feeding phase, and the oxygen level in the medium was not limited. ssDNA was isolated from reactor samples and applied on agarose gel electrophoresis. Even if the contrast was increased, weak ssDNA bands were not identifiable in samples after 2-10 h of process time. The first ssDNA band on the gel was visible in the 12 h sample. The ssDNA concentration rose to 65.02 mg L\({}^{-1}\), equivalent to 0.54 g L\({}^{-1}\) CSF1_6106 phagemid particles. The ssDNA quality was excellent during the production time, and on the agarose gel, there was only one clear band for the ssDNA product. Nevertheless, an analysis of the fraction showed that the concentration of CSF1_6106 ssDNA in the final sample is 55.33 mg L\({}^{-1}\) (STY = 2.63 mg L\({}^{-1}\) h\({}^{-1}\)), corresponding to a phagemid concentration of 0.46 g L\({}^{-1}\). The quality of the CSF1_6106 ssDNA after 21 h of process time was assessed by folding a 42-helix bundle DNA origami. Compared to the 25 L scale in Figure 3, the ssDNA concentration rose 4 h later. Therefore, the overall ssDNA concentration obtained on a 1000 L scale was lower. A possible explanation relies on the execution of the infection. On a 25 L scale, the infection material could quickly be brought into contact with the cells in a 1 L bottle by pipetting. For the 1000 L scale, the infection had to be performed in the 30 L pre-culture reactor. The volume of infection material was raised to 1 L with phosphate buffer, and the material was pumped into the headspace of the aerated stirred tank reactor by a tube connected to the lid. There may have been a loss of infection material as aerosol, which would explain the delayed ssDNA production on a 1000 L scale.