# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 3 Results  
### Variation of Infection Density  
For the coupled biomass and product formation, various multiplicities of infection (MOIs) were tested for infection one hour after initiating the batch phase. MOIs of 10\({}^{-6}\) ftu cfu\({}^{-1}\), 10\({}^{-4}\) ftu cfu\({}^{-1}\), 10\({}^{-3}\) ftu cfu\({}^{-1}\), and 10\({}^{-2}\) ftu cfu\({}^{-1}\) were applied by using pre-produced phagemid particles with CSF1_4843 as the ssDNA genome sequence. Figure 1 displays each process's cell dry weight concentration, temperature profile, feeding profile, ssDNA concentration, and agarose gels. The initial glucose was consumed after 10.6 h in all four fermentations, and the exponential feeding started. The temperature was shifted from 37 \({}^{\circ}\)C to 30 \({}^{\circ}\)C at the beginning of the feeding phase. Approximately 80 g L\({}^{-1}\) of cell dry weight was achieved without significant differences between the individual processes at the end of each process within 25-27 h.  
The variation in infection density significantly impacted the concentration of ssDNA isolated from reactor samples. Upon infection with MOI = 10\({}^{-2}\) ftu cfu\({}^{-1}\), the ssDNA concentration reached 68.73 mg L\({}^{-1}\) within 25.1 h (space-time yield STY = 2.74 mg L\({}^{-1}\) h\({}^{-1}\)). In the agarose gel electrophoresis Figure 1c, impurities, likely genomic DNA due to cell lysis, were present in the ssDNA sample after 26.6 h. This indicates that the process time for harvest is a critical factor for the product yield. Infection with an MOI of 10\({}^{-3}\) ftu cfu\({}^{-1}\) yielded 70.84 mg L\({}^{-1}\) within 27 h (STY = 2.62 mg L\({}^{-1}\) h\({}^{-1}\)), while infection with an MOI of 10\({}^{-4}\) ftu cfu\({}^{-1}\) yielded 58.71 mg L\({}^{-1}\) within 25 h (STY = 2.35 mg L\({}^{-1}\) h\({}^{-1}\)). As observed on agarose gel electrophoresis, the ssDNA quality displayed only a single product band for samples from both processes. With a comparatively low infection density of MOI = 10\({}^{-6}\) ftu cfu\({}^{-1}\), the concentration of isolated ssDNA from supernatant samples started to rise later, and the highest concentration reached was 32.1 mg L\({}^{-1}\) within 25 h (STY = 1.28 mg L\({}^{-1}\) h\({}^{-1}\)). The single-stranded DNA became visible after 19.5 h on agarose gel electrophoresis, and the ssDNA quality was rated poorly due to impurities that were detectable in the agarose gel. The ssDNA concentrations are summarized in Table 1.  
To balance process time, ssDNA quality, and ssDNA yield, we needed to find a compromise. The highest productivity was observed with a multiplicity of infection (MOI) of \(10^{-2}\) ftu cfu\({}^{-1}\), as this resulted in the greatest amount of ssDNA after 25 h. Conversely, \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Scale**} & **Multiplicity of Infection** & **CSF1\_4843 ssDNA,** & **CSF1\_6106 ssDNA,** \\  & **(MOI)** & **mg L\({}^{-1}\)** & **mg L\({}^{-1}\)** \\ \hline  & \(10^{-2}\) ftu cfu\({}^{-1}\) & 68.73 \(\pm\) 1.33 & - \\  & \(10^{-3}\) ftu cfu\({}^{-1}\) & 70.84 \(\pm\) 3.44 & 127.22 \(\pm\) 3.08 \\  & \(10^{-4}\) ftu cfu\({}^{-1}\) & 58.17 \(\pm\) 10.59 & - \\  & \(10^{-6}\) ftu cfu\({}^{-1}\) & 32.10 \(\pm\) 1.14 & - \\ \hline
25 L & \(10^{-3}\) ftu cfu\({}^{-1}\) & 191.08 \(\pm\) 2.75 & 159.10 \(\pm\) 8.44 \\ \hline
1000 L & \(10^{-3}\) ftu cfu\({}^{-1}\) & - & 55.34 \(\pm\) 3.69 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Final concentration of single-stranded DNA from high-cell-density cultivations of _Escherichia coli_ JM109 pfhp30 in 2.5 L, 25 L, and 1000 L stirred tank bioreactors infected with phagemid particles of varying genome lengths and multiplicities of infection. Figure 1: High-cell-density cultivation of _Escherichia coli_ JM109 pfhp30 in a 2.5 L stirred tank bioreactor at 37 °C with temperature shift to 30 °C at the beginning of the feeding (indicated by the vertical black line). One hour after inoculation, cells were infected with phagemid particles (CSF1\_4843) with various infection densities.