Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i>
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 hig...
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2024-12-01
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| author | Nathalie Hafner Nazanin Nematzadeh Somehsaraei Maximilian N. Honemann Hendrik Dietz Dirk Weuster-Botz |
| author_facet | Nathalie Hafner Nazanin Nematzadeh Somehsaraei Maximilian N. Honemann Hendrik Dietz Dirk Weuster-Botz |
| author_sort | Nathalie Hafner |
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| description | 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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| spelling | doaj-art-b1ca41944a0b4a9d88647ae4f5be39b32025-08-20T02:38:41ZengMDPI AGApplied Sciences2076-34172024-12-0114231145910.3390/app142311459Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i>Nathalie Hafner0Nazanin Nematzadeh Somehsaraei1Maximilian N. Honemann2Hendrik Dietz3Dirk Weuster-Botz4School of Engineering and Design, Biochemical Engineering, Technical University of Munich, 85748 Garching, GermanySchool of Engineering and Design, Biochemical Engineering, Technical University of Munich, 85748 Garching, GermanyDepartment of Physics, Biomolecular Nano-Technology, Technical University of Munich, 85748 Garching, GermanyDepartment of Physics, Biomolecular Nano-Technology, Technical University of Munich, 85748 Garching, GermanySchool of Engineering and Design, Biochemical Engineering, Technical University of Munich, 85748 Garching, GermanyLarge 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.https://www.mdpi.com/2076-3417/14/23/11459phagemid particlessDNA<i>Escherichia coli</i>helper plasmidhigh-cell-density fermentationscale-up |
| spellingShingle | Nathalie Hafner Nazanin Nematzadeh Somehsaraei Maximilian N. Honemann Hendrik Dietz Dirk Weuster-Botz Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> Applied Sciences phagemid particle ssDNA <i>Escherichia coli</i> helper plasmid high-cell-density fermentation scale-up |
| title | Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> |
| title_full | Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> |
| title_fullStr | Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> |
| title_full_unstemmed | Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> |
| title_short | Early Infection for Mass Production of Artificial Single-Stranded DNA with <i>Escherichia coli</i> |
| title_sort | early infection for mass production of artificial single stranded dna with i escherichia coli i |
| topic | phagemid particle ssDNA <i>Escherichia coli</i> helper plasmid high-cell-density fermentation scale-up |
| url | https://www.mdpi.com/2076-3417/14/23/11459 |
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