# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 1 Introduction
This so-called reverse infection method combines a designed phagemid particle and an optimized helper plasmid. The phagemid codes for gene 3 of the M13 bacteriophage, the phage ori, a packaging signal, and a freely chosen custom target sequence. The helper plasmid provides the other genes of the M13 phage in an optimized arrangement. Up to 83 mg L\({}^{-1}\) ssDNA was obtained in a stirred tank bioreactor, realizing up to a 40-fold increase in product concentration over previous phage-free ssDNA production methods [27; 28]. Biomass and product formation were separated in the established high-cell-density fermentation process. The positive effect of decoupling biomass and product formation has been demonstrated for M13 phages [26]. Additionally, bacterial cells infected with M13 phages stop phage production after a while because, in the first several generations, the level of free p5 is built up. This protein contributes to increased sequestration and packaging of newly synthesized ssDNA rather than its conversion to replicative form (RF) DNA. Simulations revealed that the RF DNA in the cell is constant for seven generations and decreases then with each subsequent cell division. Nearly 80% of the total phages produced have been extruded from the cell by the seventh generation. Curing occurs when less than one copy of M13 RF DNA is present in the cell [29]. Cells become infected when a high number of _E. coli_ cells have already been produced. Five hours after initiation of the glucose feeding, the _E. coli_ cells were infected with phagemid particles with a multiplicity of infection (MOI) of one transforming unit per colony-forming unit (tfu cfu\({}^{-1}\)). This corresponds to a phagemid particle concentration in the bioreactor of 1 mg L\({}^{-1}\). The current method requires producing a large amount of infection material in advance. Batch bioreactor cultivations enable concentrations of 2-3 mg L\({}^{-1}\) circular ssDNA within 12 h of process time [27]. Consequently, pre-produced phagemid particles from a one-liter batch cultivation would be necessary to infect around 13 L of _E. coli_ cells at an optical density (OD\({}_{600}\)) of 50 with a multiplicity of infection of 1 ftu cfu\({}^{-1}\). This effort for phagemid particle pre-production might be feasible on a small scale but not practical for larger scales. In this study, we aimed to reduce the required amount of infection material by linking cell growth with phagemid particle production. This was realized by infecting only a few cells at the beginning of the batch phase of the high-cell-density fermentation process. Initially infected cells start the production of new phagemid particles, which can again infect other cells. This leads to a fluent infection of all cells in the bioreactor during the growth of _E. coli_. Different multiplicities of infection (MOIs) were applied using pre-produced phagemid particles to identify an ideal amount of infection material for the early process stage. The best-performing early infection process was transferred to a 25 L scale, and a 1000 L scale to study the scalability of ssDNA production.