monarch pcr dna cleanup spin columns  (New England Biolabs)

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

Article Snippet: Initial purification of the PCR amplicons was conducted using Monarch PCR & DNA Cleanup spin columns (New England Biolabs, Ipswitch, MA).

Techniques: Generated, Transformation Assay, Electroporation, Isolation, Variant Assay, Luciferase, Infection, Lysis, Bacteria

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

Article Snippet: Initial purification of the PCR amplicons was conducted using Monarch PCR & DNA Cleanup spin columns (New England Biolabs, Ipswitch, MA).

Techniques: Modification, Amplification, Size Selection, Nucleic Acid Purification, Biomarker Discovery, Nucleic Acid Electrophoresis, Electroporation, Isolation, Sequencing