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

Journal: Nature Communications

Article Title: A family of NADPH/NADP + biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes

doi: 10.1038/s41467-024-55302-x

Figure Lengend Snippet: a Diagram showing the development of selected NAD and NADP sensors including NAPstars. b AlphaFold2 prediction of NAPstar structure. Graphs showing the normalised logarithm of the cpT-Sapphire/mCherry fluorescence ratio at c different NADPH/NADP + and d NADH/NAD + ratios. NADPH and NADH concentration was titrated against a fixed background of 150 µM NADP + and 500 µM NAD + respectively. In c and d , the dashed lines show a fitted sigmoidal function that was used to determine K d(NAD(P)H) . e Table summarising the determined K d(NADPH) and K d(NADH) values of all NAPstars. Graphs showing the normalised logarithm of the cpT-Sapphire/mCherry fluorescence ratio at f different NADPH/NADP + and g NADH/NAD + ratios. NADP + and NAD + concentration was titrated against a fixed concentration of NADPH and NADH respectively that for each probe corresponded approximately to the determined K d(NADPH) and K d(NADH) values. This experimental regime, by definition, only allows a maximum of approximately 50% NADPH binding and explains the difference in shape of the titration curve between panels c and f . h Table summarising the determined K r(NADPH/NADP+) for all NAPstars. For panels c , d , f , g , n = 3 technical replicates. Data are presented as mean ± s.d. normalised to the lowest data point.

Article Snippet: All coding sequences for NAPstars, Peredox, HyPer7 and NERNST were codon optimised for expression in Saccharomyces cerevisiae , synthesised and delivered in a pUC57 plasmid (GenScript Biotech, Rijswijk, Netherlands).

Techniques: Fluorescence, Concentration Assay, Binding Assay, Titration

Journal: Nature Communications

Article Title: A family of NADPH/NADP + biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes

doi: 10.1038/s41467-024-55302-x

Figure Lengend Snippet: a Photograph of the coupled fermentor–fluorimeter setup used to monitor redox changes in YMC-synchronised cultures. b Diagram illustrating the coupled metabolic and cell division cycles observed during the YMC. CDC (cell division cycle), HOC (high oxygen consumption), and LOC (low oxygen consumption). c Representative traces showing the changes in dissolved oxygen, NAPstar4.3 (NADP redox state), Peredox (NAD redox state), and Hyper7 (H 2 O 2 ) during two complete cycles of the YMC ( n = 2, in which probe dynamics were measured for multiple YMC cycles in two independent YMC-synchronised cultures; Supplementary Fig. ).

Article Snippet: All coding sequences for NAPstars, Peredox, HyPer7 and NERNST were codon optimised for expression in Saccharomyces cerevisiae , synthesised and delivered in a pUC57 plasmid (GenScript Biotech, Rijswijk, Netherlands).

Techniques:

Journal: Nature Communications

Article Title: A family of NADPH/NADP + biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes

doi: 10.1038/s41467-024-55302-x

Figure Lengend Snippet: a Confocal microscopy images of NAPstar4.3 expressed in the cytosol of Arabidopsis thaliana plants. Scale bar = 20 µm. Response of NAPstar4.3 ( b ), NAPstarC ( c ) and Peredox ( d ) to the indicated periods of illumination after treatment with a solvent control (Mock) or the photosynthetic inhibitor DCMU (in each panel data presented are the mean ± s.d. based on n = 6 leaf discs from six individual plants). e , Box and whisker plot, derived from the datasets in b – d , showing the change in the normalised log10 TS/mC ratio after 60 minutes of illumination. Boxes show the interquartile range, with the middle line defining the median. X, represents the mean values. Whiskers show the minimum and maximum values, excluding outliers. Dots indicate outlier values, which are defined as being 1.5 times the interquartile range above and below the third and first quartile respectively. P -values are derived from an unpaired two-tailed Student’s t -test. Response of NAPstar4.3 ( f ), NAPstarC ( g ), Peredox ( h ), Grx1-roGFP2 ( i ) and roGFP2-Orp1 ( j ) probes to 6 hours of hypoxia (0.1% oxygen) followed by restoration of normal atmospheric oxygen levels ( n = 7 for NAPstarC, roGFP2-Orp1 and Grx1-roGFP2, n = 8 for Peredox and NAPstar4.3leaf discs taken from 7 or 8 individual plants). In all panels, data are presented as mean ± s.d. normalised to the average value before induction of hypoxia.

Article Snippet: All coding sequences for NAPstars, Peredox, HyPer7 and NERNST were codon optimised for expression in Saccharomyces cerevisiae , synthesised and delivered in a pUC57 plasmid (GenScript Biotech, Rijswijk, Netherlands).

Techniques: Confocal Microscopy, Solvent, Control, Whisker Assay, Derivative Assay, Two Tailed Test