Journal: bioRxiv

Article Title: An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

doi: 10.1101/2021.01.30.428974

Figure Lengend Snippet: The MPyV nanocompartment platform for yeast. (a) MPyV virus-like particles (VLPs) are formed by the self-assembly of two protein components, VP1 (wt or an NLS-deletion mutant, Δ) and VP2C linked to the cargo protein of interest (‘POI’). (b) Transmission electron micrographs of purified VLPs expressed in the absence and presence VP2C-GFP.

Article Snippet: ΔVP1 was codon-optimised for yeast and synthesised by GenScript.

Techniques: Virus, Mutagenesis, Transmission Assay, Purification

Journal: bioRxiv

Article Title: An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

doi: 10.1101/2021.01.30.428974

Figure Lengend Snippet: VLP characterisation. (a) SDS-PAGE gel of purified VLP samples stained with Coomassie blue. Arrows show the position of VP1 and cargo bands. ‘MW’ = protein molecular weight marker. (b) Native gel electrophoresis of purified particles. Samples (3 μg) were loaded on a 1% agarose gel alongside 0.5 μg of a DNA molecular ladder (lane L). GFP signal from intact particles can be visualised with blue light illumination and a 530 nm emission filter. Nucleic acid and protein were stained with GelRed and Coomassie blue respectively. (c) Particle size distributions, measured with nanoparticle tracking analysis (NTA). The mode and width at half height (W0.5) of each distribution is indicated. (d) Changes in size distribution and molar mass of wtVP1 and ΔVP1 VLPs with GFP loading, as determined by SEC-MALS. Refractive index, RI (normalised to the mode) is shown as lines and molar mass is shown as circles. The dashed light grey line indicates the theoretical mass of empty VLPs corresponding to each VP1 variant.

Article Snippet: ΔVP1 was codon-optimised for yeast and synthesised by GenScript.

Techniques: SDS Page, Purification, Staining, Molecular Weight, Marker, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Refractive Index, Variant Assay

Journal: bioRxiv

Article Title: An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

doi: 10.1101/2021.01.30.428974

Figure Lengend Snippet: Growth profiles and protein expression levels of MIOX-expressing strains. (a) Cell density (OD 600 ) against time post-induction. (b) GFP fluorescence was tracked by flow cytometry as a proxy for MIOX levels in the three GFP-tagged MIOX constructs. (c) Anti-GFP and anti-VP1 western blots of cell lysates at 24 h and 72 h post-induction. The same amount of cells was loaded per lane, based on the OD 600 reading. Bands on the anti-GFP blot match the expected size of each corresponding MIOX fusion protein. (d) MIOX compartments isolated by iodixanol cushion ultracentrifugation, negatively stained and viewed under TEM. All data points in (a) and (b) are the means of 3 biological replicates; error bars are +/− 1 STD.

Article Snippet: ΔVP1 was codon-optimised for yeast and synthesised by GenScript.

Techniques: Expressing, Fluorescence, Flow Cytometry, Construct, Western Blot, Isolation, Staining