sanger dna sequencing  (Azenta)

Journal: Nature Communications

Article Title: Engineering plasmids with synthetic origins of replication

doi: 10.1038/s41467-026-68907-1

Figure Lengend Snippet: A Schematic of the MB1 plasmid origin of replication (ORI) mechanism. The RNA primer is transcribed by RNA polymerase (RNAP) and, by default, folds into an RNA structure that is processed by RNase H to create a substrate for DNA polymerase I (Pol I) to initiate DNA replication. The antisense RNA is produced from an overlapping gene, which, if allowed to interact with the RNA primer, induces an alternative fold that does not initiate DNA replication. As the concentration of antisense RNA is proportional to the plasmid copy, this serves as a copy-control negative feedback loop. B Schematic of a refactored pMB1 ORI that separates the RNA primer and antisense RNA gene and introduces inactivating mutations in the P1 promoter sequence. C Shows sequences of mutations to inactivate the P1 promoter encoded inside the RNA primer. (Left graph) Whole-cell fluorescent characterization of the mutant promoters using a fluorescent reporter gene. (Right graph) Relative copy number of plasmids containing the original pMB1 ORI or refactored ORI with an RNA primer containing mutant P1 promoters. Copy number was characterized by encoding a constitutive RFP expression cassette onto the plasmid. Fluorescence characterization was performed (measured in units of fluorescence/optical density (OD) at 600 nm) in E. coli cells. Data show mean values ± SD and individual values of n = 4 biological replicates. Source data for this figure is available in the Source Data file.

Article Snippet: All assembled plasmids were verified using Sanger DNA sequencing (Genewiz) or Nanopore sequencing (Plasmidsaurus).

Techniques: Plasmid Preparation, Produced, Concentration Assay, Control, Sequencing, Mutagenesis, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Engineering plasmids with synthetic origins of replication

doi: 10.1038/s41467-026-68907-1

Figure Lengend Snippet: A SynORI plasmids (pSynORI) can be programmed to convert distinct chemical signals into DNA copy output using transcriptional regulators to control RNA primer transcription. Relationship between relative copy number and chemical input for pSynORI built using (B) Plac (activated by IPTG), C PcymR (activated by cumate), D the ZTP riboswitch (activated by Z), E and the yxjA riboswitch (repressed by 2AP). F Matrix showing the relative copy of a pSynORI that is activated by cumate and repressed by IPTG with inducer titrations. Relative copy number was characterized by encoding a constitutive RFP expression cassette onto the plasmid in E. coli cells (measured in units of fluorescence/optical density [OD] at 600 nm). G Schematic of compatible and inducible pSynORI that converts chemical input signals into a DNA copy, which can be read using sequencing. A pSC101 plasmid with a non-inducible copy number is included as a reference. H The relative copy of pSynORI A and B was calculated using the reference plasmid. Bar plots show the DNA copy ratio of pSynORI A and B. Data in B – E and F shows n = 4 biological replicates, and Data in H show n = 3 biological replicates. All error bands indicate SD. Source data for this figure is available in the Source Data file.

Article Snippet: All assembled plasmids were verified using Sanger DNA sequencing (Genewiz) or Nanopore sequencing (Plasmidsaurus).

Techniques: Control, Expressing, Plasmid Preparation, Fluorescence, Sequencing