Phage‐free production of artificial ssDNA with Escherichia coli

Abstract

Artificial single‐stranded DNA (ssDNA) with user‐defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass‐producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross‐contaminating manufacturer plants with self‐replicating phages. Here we overcome this problem with an efficient, scalable, and cross‐contamination‐free method for the phage‐free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self‐replication in the absence of the helper plasmid. This enables cross‐contamination‐free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by‐products and increased the maximal product concentration up to 83 mg L−1 of ssDNA in a stirred‐tank bioreactor, thus realizing up to a 40‐fold increase in maximal product concentration over previous scalable phage‐free ssDNA production methods.

Circular Single-Stranded DNA: Discovery, Biological Effects, and Applications

“…Both genomes contain an f1 single-stranded ori without a double-stranded ori, effectively avoiding dsDNA contaminations (Figure B). The subsequent period did not witness any significant advancements in the literature about the synthesis of CssDNA by bacteriophages. ,− …”

Biotechnological Frontiers of DNA Nanomaterials Continue to Expand: Bacterial Infection using Virus‐Inspired Capsids

“…This could be either loaded as cargo, or perhaps be a tool for the “undressing ”of the staples to release the scaffold constitutively present in the DNA origami particle, and liberate it as a template for reproduction. To control or reduce infectivity, a split plasmid approach, as recently discussed by Behler et al., [9] could be incorporated. Lastly, with bacterial delivery being a fresh target of DNA nanotechnology, many fundamental parameters including size, stability and geometry of the DNA‐based architecture, which likely affect uptake yield and kinetics, are left to be explored.…”

Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography

“…Although both methods are well-established and are easy-to-apply, the results obtained are often inaccurate due to a lack of selectivity due to the presence of impurities. When Escherichia coli/phage systems are used to produce ssDNA, phage-derived impurities like proteins, and cell-derived impurities like genomic DNA and cell debris, will increase absorbance at 260 nm, thus contributing to overestimate the scaffold concentration. , On the other hand, the use of densitometry analysis of DNA bands in agarose gels will typically underestimate the ssDNA concentration since the commonly used dye molecules for gel staining intercalate less on ssDNA compared to double-stranded DNA (dsDNA) . While different fluorescent dyes can be used to quantify ssDNA with high sensitivity (1 ng/mL), they lack sensibility and present fluorescence enhancement when bound to dsDNA and RNA.…”