Chemostat studies of bacteriophage M13 infected Escherichia coli JM109 for continuous ssDNA production

Kick, Benjamin; Behler, Karl L.; Severin, Timm Steffen; Weuster‐Botz, Dirk

doi:10.1016/j.jbiotec.2017.06.409

Cited by 4 publications

(7 citation statements)

References 28 publications

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“…This separation allows the microorganisms to grow to high cell density without the metabolic burden of product formation. For M13 phages especially the positive effect of the decoupling has been shown (Kick, Behler, et al, 2017). Additionally, bacterial cells infected with M13 phages stop phage production after a while (Smeal et al, 2017).…”

Section: Resultsmentioning

confidence: 93%

“…The handling of the samples changes throughout the process due to the increase in viscosity. In the biotechnological process for high cell density production of M13‐genome derived ssDNA, Kick, Behler, et al (2017) show an optimal harvesting time after about 30 h of feeding. Here we recommend harvesting after 16–17 h of feeding as viscosity is still low enough for the downstream processing and production of ssDNA is almost at an end.…”

Section: Resultsmentioning

confidence: 99%

“…The handling of the samples changes throughout the process due to the increase in viscosity. In the biotechnological process for high cell density production of M13-genome derived ssDNA, Kick, Behler, et al (2017) show an optimal harvesting time after about 30 h of feeding.…”

Section: Production Process Adaptionsmentioning

confidence: 99%

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Phage‐free production of artificial ssDNA with Escherichia coli

Behler

Honemann

Silva-Santos

et al. 2022

Biotech & Bioengineering

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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.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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Phage‐free production of artificial ssDNA with Escherichia coli

Behler

Honemann

Silva-Santos

et al. 2022

Biotech & Bioengineering

View full text Add to dashboard Cite

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“…The production of scaffold DNA for DNA origami manufacturing is usually performed by the purification of phage-derived single-stranded genomic DNA , or with asymmetric PCR (aPCR) from DNA templates. , Scaffold quantification is typically based on absorbance at 260 nm or on agarose gel electrophoresis densitometry. 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.…”

Section: Introductionmentioning

confidence: 99%

“…The phage expansion occurred overnight under the same conditions. Phage purification and genomic ssDNA extraction were performed according to Kick et al 11 aPCR Mixtures. The target 449 and 1000 nt ssDNA scaffolds were synthesized by aPCR with different ratios of forward to reverse primers.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Silva-Santos,

Sousa Rosa,

Marques

et al. 2024

ACS Omega

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DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

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