Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i>
2024
Nathalie Hafner | Nazanin Nematzadeh Somehsaraei | Maximilian N. Honemann | Hendrik Dietz | Dirk Weuster-Botz
Large quantities of artificial single-stranded DNA (ssDNA) with user-defined sequences are increasingly required to exploit the potential of DNA nanotechnology. Cross-contamination-free ssDNA production can be achieved using <i>Escherichia coli</i> with an optimized helper plasmid in high-cell-density cultivation via the secretion of phagemid particles containing ssDNA with user-defined sequences. In our study, we aimed to reduce the number of phagemid particles for the initiation of ssDNA production. We tested different infection densities, ranging from a multiplicity of infection (MOI) of 10<sup>−6</sup>–10<sup>−2</sup> tfu cfu<sup>−1</sup> at the start of the initial batch phase in a 2.5 L stirred tank bioreactor. A MOI of 10<sup>−3</sup> tfu cfu<sup>−1</sup> was the best compromise between process time and ssDNA concentration. Early initiation of ssDNA production with low MOI reduced the number of phagemid particles by a factor of 250,000. The early infection strategy was successfully scaled up to the 25 L scale, resulting in ssDNA concentrations of >100 mg L<sup>−1</sup> within a process time of one day. Transferring the infection strategy to a 1000 L scale gained 65 mg L<sup>−1</sup> ssDNA because of incomplete initial infection. The versatility of the early infection strategy was further proven with a second prolonged, user-defined ssDNA sequence.
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