# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 4 Discussion  
The efficient and scalable production of artificial ssDNA with _E. coli_ has been improved by reducing infection material by a factor of 250,000 from 1 mg L\({}^{-1}\) to 4 ng L\({}^{-1}\). Cell growth and product formation were coupled to realize the reverse infection with only a fraction of the infection material. This infection strategy is more economical than earlier infection strategies [28]. with decoupling of cell growth and a phagemid particle production phase. As batch cultivation in shake flasks or stirred tank bioreactors for phagemid particle pre-production has low yields [27], the phagemid particle pre-production works only on a laboratory scale but is elaborated and not feasible on large scales. If infection of _E. coli_ cells at OD\({}_{600}\) = 50 with an infection density of MOI = 1 ftu cfu\({}^{-1}\) is applied and the number of phagemid particles corresponding to an ssDNA concentration of 2 mg L\({}^{-1}\) in the pre-production is assumed, around 59 L of pre-produced material would be required to infect a reactor volume of 750 L. With the same assumptions, our new infection strategy requires only 235 uL phagemid particle pre-production for infection at OD\({}_{600}\) = 0.2 with an infection density of MOI = 10\({}^{-3}\) ftu cfu\({}^{-1}\). The new infection strategy produced 191.08 mg L\({}^{-1}\) CSF1_4843 ssDNA on a 25 L scale. Compared to the previously published concentration of 83 mg L\({}^{-1}\) in a separated biomass and product formation [28], a 2.3-fold increased amount of ssDNA was produced in this study. The ssDNA concentrations are comparable to prior published biotechnological mass production of DNA origami by [PERSON] et al. in laboratory-scale stirred tank bioreactor. Contaminations of helper-phage ssDNA in the product are precluded by design in our system [37]. A more extended custom sequence of ssDNA was produced biotechnologically using the new early infection method. The genome size of M13 phages can range from 4-12 Kbp [38]. CSF1_6106 is 1.26 times longer than CSF1_4843, which is still half the possible genome length. The _Escherichia coli_ growth was influenced by the more extended phagemid version, so 38% less biomass was produced than in the 4843 bp ssDNA production. It is already known that after phage infection, _E. coli_ experiences envelope stress due to the large envelope channel and a hugely increased membrane protein production, resulting in slow growth [39]. CSF1_6106 results in double the product concentration within less time compared to CSF1_4843. An influence on the _E. coli_ cells by the higher burden of phagemid particle production is conceivable. The ssDNA concentration with the extended phagemid version CSF1_6106 was improved on a 2.5 L scale, while on a 25 L scale, a slide decrease in product formation was recognized compared to the shorter version. The process was transferred to a 1000 L scale for the first time. The production of phagemid particles and, thus, ssDNA on this scale was 4 h delayed compared to the 25 L stirred tank bioreactor, which resulted in a lower final ssDNA concentration. The infection was conducted differently on the 25 L and 1000 L scales. In both cases, phagemid particles were added to the cells at the end of the second pre-culture. On a 25 L scale, the cells intended for inoculation were collected in a 1 L bottle, and the phagemid particles as infection material were transferred aseptically directly to the cell suspension using a pipette, before the infected cell suspension was transferred to inoculate the stirred tank bioreactor. In contrast, on a 1000 L scale, the infection was performed at the end of the second pre-culture stage by transferring the infection material to the 50 L stirred tank bioreactor. The volume was increased to minimize potential material loss during this transfer, e.g., liquid leavings in the transfer line, connectors, or the container.