# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 3 Results
### Phagemid Elongation  
In the next step, we created a more extended version of the CSF1 phagemid, termed CSF1_6106, to investigate the impact of phagemid length on the ssDNA production. Phagemid particles were produced in shaking flasks in advance, and the cells were infected with MOI = 10\({}^{-3}\) ftu cfu\({}^{-1}\) at the beginning of the batch phase. By infecting the cells just before transferring them to the main culture, we ensured that the cells and infection material came into contact with each other and reduced the loss of infection material through discharge into the headspace of the gassed stirred tank bioreactor.  
Figure 2 shows cell dry weight concentration, temperature, feeding profile, and ssDNA concentration against the process time and the agarose gel electrophoresis of ssDNA isolated from the samples.  
Infection of _E. coli_ JM109 cells with CSF1_6106 phagemid particles negatively influenced cell growth. The highest cell dry weight concentration was observed in the 23 h sample with 49.58 g L\({}^{-1}\), with a following decrease. Cells were removed from samples, and the supernatant was used to isolate ssDNA. The ssDNA concentration curve followed a linear increase between 12 and 21 h of process time. An ssDNA concentration of 143.10 mg L\({}^{-1}\) was measured within 21 h, equivalent to 1.18 g L\({}^{-1}\) phagemid particles. Considering the fraction estimation in this sample, 127.22 mg L\({}^{-1}\) was measured (Table 1) (STY = 6.06 mg L\({}^{-1}\) h\({}^{-1}\)), equivalent to 1.05 g L\({}^{-1}\) phagemid particles. After 21 h, the concentration curve for the overall measured concentration still rose, while the ssDNA concentration curve corrected by fraction estimation stopped r  
Figure 2: High-cell-density cultivation of _Escherichia coli_ JM109 pfhp30 in a 2.5 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 phagemid particles (CSF1_6106) with MOI = 10\({}^{-3}\) ftu cfu\({}^{-1}\). Error bars refer to the standard deviations of triplicates in measurements. (**a**) Cell dry weight concentration, temperature, and feed profile. (**b**) ssDNA concentration was calculated from the absorbance at 260 nm (black circles). Then, samples were analyzed using agarose gel electrophoresis to estimate the fraction of actual product ssDNA using ImageJLab (gray circles). (**c**) ACEP of the ssDNA (scaffold) produced in this cultivation.  
level. The agarose gel electrophoresis showed clear product bands, indicating good product quality. Impurities of other DNA fragments occurred between 23 and 26 h. This explains the rising ssDNA concentration for the overall measurement at 260 nm, while the corrected concentration by fraction estimation was not increasing anymore. This suggests an optimal harvest time after only 21 h of process time because, after that, the amount of impurities rises faster than the product concentration.  
CSF1_6106 is 1.26 times longer than CSF1_4843, but it results in double the product concentration within less time. This shows that longer custom-sequence ssDNA molecules can be produced biotechnology using early infection.