ms2 vlp expression plasmid backbone  (Addgene inc)

Journal: Nature Communications

Article Title: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

doi: 10.1038/s41467-020-18130-3

Figure Lengend Snippet: a Schematic of the genetic construct of the engineered MS2-SARS-CoV-2 N-gene VLP encompassing the MS2 maturation protein (MP) in green and coat proteins (CP) in orange, linked via His-tag (yellow), under transcriptional control of the T7 promoter and T7 terminator sequences. The SARS-CoV-2 N protein RNA is packaged using a downstream pac site . Schematic created using DNAplotlib . b The VLP constructs were expressed in E. coli and purified using HiTrap® TALON® Crude and HiTrap® Heparin columns. SDS-PAGE analysis of the purification steps includes a protein marker (M) followed by pellet (P) and soluble fraction (S) of the cell lysate, followed by the column flow through (FT) and protein elution fractions, where the CP-His-CP dimer (~28 kDa) is indicated by an arrow. c Purified VLPs were analysed by dynamic light scattering (DLS), which showed a uniform particle population of ~27 nm. Error bars represent the SD of three technical replicates. d Reverse-transcriptase droplet digital PCR (RT-ddPCR) was performed for absolute quantification of the purified VLPs. Serial dilutions of 1, 10, and 100 thousand-fold of the purified VLPs in the presence and absence of a reverse transcription (RT) enzyme were analysed. Droplets were clustered using a threshold determined using a python implementation of an online tool ( http://definetherain.org.uk ). Dotted lines represent the cut offs for the positive and negative clusters. Any data points between the two dotted lines are considered droplet “rain”. Source data are available in the Source Data file.

Article Snippet: The N-gene was cloned into a MS2 VLP expression plasmid backbone (Addgene #128233) using Type IIs assembly.

Techniques: Construct, Control, Purification, SDS Page, Marker, Reverse Transcription, Digital PCR, Quantitative Proteomics

Journal: Nature Communications

Article Title: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

doi: 10.1038/s41467-020-18130-3

Figure Lengend Snippet: a Schematic of the CRISPR-Cas13a nucleic acid detection workflow from patient samples. Viral RNA is amplified using target-specific primers and detected with target-specific crRNA activating Cas13a to collaterally cleave a fluorescent probe. b CDC N1, N2, and N3 primer sets were employed to amplify the N-gene RNA released from MS2-SARS-CoV-2 VLPs at 2.5, 25, and 250 copies per reaction. The CRISPR-Cas13a detection time course was analysed using a fluorescence microplate reader. Error bars represent the SEM of n = 3 biologically independent amplification reactions and four CRISPR detection technical replicates. c Schematic of the RT-loop-mediated isothermal amplification (RT-LAMP) diagnostic workflow using target-specific LAMP primers. The isothermal amplification of a target results in the acidification of the RT-LAMP master mix and a subsequent pH-associated colour change that is detected using a microplate reader. d Time-course detection of three replicate RT-LAMP reactions using the MS2-SARS-CoV-2 VLPs at 0, 5, 10, 20, 30, and 40 copies per reaction, performed at 65 °C using the BMG CLARIOstar Plus microplate reader. Source data are available in the Source Data file.

Article Snippet: The N-gene was cloned into a MS2 VLP expression plasmid backbone (Addgene #128233) using Type IIs assembly.

Techniques: CRISPR, Amplification, Fluorescence, Diagnostic Assay