# Early Infection for Mass Production of Artificial Single-Stranded DNA with _Escherichia coli_
## 1 Introduction  
DNA is typically double-stranded in living organisms, whereas single-stranded DNA (ssDNA) occurs only in viruses or in a transient state during the replication of double-stranded DNA (dsDNA). Long ssDNA has different applications in genome editing and DNA nanotechnology. It has been shown that genome editing using CRISPR/Cas is significantly more efficient when ssDNA is used as a so-called donor for the repair mechanism instead of double-stranded DNA [1, 2]. In nanotechnology, DNA origami is a method to construct user-defined three-dimensional (3D) nanoscale structures enabling precise control of size, shape, and composition [3]. Long single-stranded DNA scaffolds serve as building material for assembly with a set of oligonucleotides called staples, using Watson-Crick base pairing to fold into predefined shapes [4, 5]. The self-assembly can proceed rapidly and with high yields and quality, depending on the design and assembly procedures used [6]. Multiple discrete DNA origami can oligomerize into well-defined higher-order 3D objects with dimensions that can exceed those of viruses. DNA origami nanoparticles are currently being explored for applications such as drug delivery systems, biosensing, and molecular motors or as direct therapeutic agents for virus neutralization [3, 7, 8, 9, 10, 11, 12]. Large quantities of artificial single-stranded DNA (ssDNA) will be increasingly required to exploit the potential of these and other future applications. The ssDNA production can be performed by chemical synthesis or various enzymatic methods. For oligonucleotidesotides (10-100 nt of the length), phosphoramidite-based chemical synthesis is the method of choice with its low cost, high speed, and high efficiency [13; 14]. Due to an increasing error rate with increasing length, fragments longer than 150 nt are difficult to synthesize [15; 16]. Enzymatic methods, like rolling circle amplification (RCA) require pure components like DNA polymerase, a circular ssDNA template, short ssDNA primer, and dNTPs. RCA can synthesize long ssDNA molecules, but their yield is limited to a few milligrams [17]. Another enzymatic method, the asymmetric polymerase chain reaction, succeeded in the production of 690 ng ssDNA per 50 \(\mu\)L reaction volume with up to 15 kb length [18; 19]. Other ssDNA production methods exist, like strand-specific lambda exonuclease digestion [20] or PCR with biotinylated 3\({}^{\prime}\) primers followed by strand separation via streptavidin-coated magnetic beads [21; 22]. All these methods can be costly and difficult to scale up. An alternative and more scalable solution for ssDNA production uses bacteriophages with fast-growing _Escherichia coli_ (_E. coli_) cells that carry the F-episome as hosts. The filamentous, nonlytic bacteriophage M13 comprises a circular 6400 nucleotide ssDNA genome enveloped by approximately 2700 copies of a major coat protein (p8) and five copies of each minor coat protein (p3 and p6 on one side and p7 and p9 on the other side). The genome encodes 11 genes in two transcriptional units. An intergenic region contains the replication origins and a packaging signal required for particle formation [23]. The viral ssDNA is amplified intracellularly, and phages are assembled simultaneously to release without lysis of the host cells [24]. [PERSON] et al. developed a high-cell-density fermentation process of bacteriophage-infected _E. coli_ in a stirred tank bioreactor under controlled pH, dissolved oxygen, and substrate supply conditions, obtaining up to 0.41 g of purified single-stranded scaffold DNA per liter of fermentation broth [25]. Feeding optimization achieved an ssDNA space-time yield of 5.7 mg L\({}^{-1}\) h\({}^{-1}\)[26]. As self-replicating particles, phages lead to a risk of cross-contamination for manufacturer plants. To overcome this problem, a cross-contamination-free method was developed for the biotechnological production of artificial ssDNA with _Escherichia coli_.