# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 3 Results
### Transfer to a 25 L Scale  
The infection strategy with phagemid particles CSF1_4843 and CSF1_6106, was transferred to a 25 L scale. Figure 3 shows cell dry weight concentration, temperature, feeding profile, and ssDNA concentration against the process time and agarose gel electrophoresis of ssDNA isolated from samples of both cultivations. The initial glucose (20 g L\({}^{-1}\)) was consumed after 8.18 h (cultivation infected with CSF1_4843 phagemid particles) and 8.53 h (cultivation infected with CSF1_6106 phagemid particles), respectively. During the feeding phase, glucose was pumped into the stirred tank bioreactor according to an exponential profile. After 9.8 h, the feeding was lowered to constant pump rates to prevent oxygen limitation of the _E. coli_ cells. With the exponential feeding, the cell dry weight concentration also rose exponentially. The rise in the curve flattened when the feeding was lowered to constant rates. Independent of the infection material, the cultivations showed a reduced cell dry weight concentration of 50.6 g L\({}^{-1}\) (infection with CSF1_4843) and 45.5 g L\({}^{-1}\) infection with CSF1_6106). The reduced cell dry weight concentration was consistent with the cultivation infected with CSF1_6106 shown in Figure 2. The cell dry weight concentration was much lower than in the cultivations in Figure 1, infected with CSF1_4843 phagemid particles.  
ssDNA was isolated from reactor samples and applied on agarose gel electrophoresis. The gels showed the first visible ssDNA band in the 8 h sample. For the CSF1_6106 ssDNA, the band in the eighth- and tenth-hour samples had a low intensity. However, the weak bands could be made visible by increasing the contrast in the gel picture. The ssDNA concentrations in both cultivations rose and began to reach a plateau between 23.5 and 25 h. At the end of the process, after 25 h, 262.84 mg L\({}^{-1}\) CSF1_4843 ssDNA, equivalent to 2.17 g L\({}^{-1}\) CSF1_4843 phagemid particles, were obtained. Considering the fraction estimation in this sample, 191.08 mg L\({}^{-1}\) was measured (STY = 7.64 mg L\({}^{-1}\) h\({}^{-1}\)), equivalent to 1.58 g L\({}^{-1}\) phagemid particles. The cultivation infected with CSF1_6106 resulted in an ssDNA concentration of 239.97 mg L\({}^{-1}\) and 1.98 g L\({}^{-1}\) of phagemid particles. When multiplied by the percentage of the fraction estimation, a concentration of 159.10 mg L\({}^{-1}\) CSF1_6106 ssDNA (STY = 6.36 mg L\({}^{-1}\) h\({}^{-1}\)), corresponding to 1.31 g L\({}^{-1}\) CSF1_6106 phagemid particles, was obtained. The ssDNA concentrations are summarized in Table 1. The applications of the ssDNA samples from both cultivations on agarose gel electrophoresis indicated the occurrence of impurities as smears on the gel after 22 h of process time. The quality of the ssDNA after 25 h of process time was assessed by folding of 42-helix bundle DNA origamis for both scaffolds.  
The process data on a 25 L scale were comparable, independent of the phagemid length. In the case of infection with CSF1_6106, the cell dry weight concentration and growth of the _E. coli_ cells is comparable to the 2.5 L scale. This is not the case for cultivations infected with CSF1_4843 phagemid particles, where a much higher cell dry weight concentration was obtained on a 2.5 L scale compared to a 25 L scale. The CSF1_4843 ssDNA concentration isolated from samples of the 25 L scale cultivation was 3.7 times higher than on the 2.5 L scale, whereas the CSF1_6106 ssDNA concentration was 1.7 times higher than on the 2.5 L scale.