MOESM2 of Engineering xylose metabolism in thraustochytrid T18
Creators: name: Merkx-Jacques, Alexandra
name: Rasmussen, Holly
name: Muise, Denise
name: Benjamin, Jeremy
name: Haila Kottwitz
name: Tanner, Kaitlyn
name: Milway, Michael
name: Purdue, Laura
name: Scaife, Mark
name: Armenta, Roberto
name: Woodhall, David
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: Cardiology

description: Additional file 2: Figure S1. Glucose (Glc) and xylose (Xyl) consumption by wild-type over time and xylitol (Xyt) identification. A) Chromatograms of the culture supernatant on days 0 (dashed red line), 1, 2, 3 (solid black line), 4 (dashed cyan line), and 7 (solid blue line). B) Chromatograms of the culture supernatant from day 7 before (solid blue line) and after (dashed line) spiking with xylitol. C) Mass spectrometry analysis of the unknown peak. Figure S2. SDS-PAGE of metal chelation fractions used for in vitro protein activity assays. Lane 1: molecular weight marker, lane 2: fraction containing histidine-tagged putative xylose isomerase, lane 3: fraction containing mostly the co-eluting band. Figure S3. Effect of temperature on (A) XylA and (B) T18 XI activity on xylose and xylulose. The mean is plotted with the error bars representing the highest and lowest values of duplicate assays. Symbols: diamonds, xylose; squares, xylulose. Figure S4. Dose dependence of (A) XylA and (B) T18 XI with xylose (diamonds) or xylulose (squares). A negative control with fraction containing the 39 kDa co-eluted protein but undetectable levels of 52 kDa protein is also shown (filled symbols). XylA assays were done in duplicate at 30°C; the mean is plotted with the error bars representing the highest and lowest values. T18 xylose isomerase assays were done in triplicate at 50°C, the mean is plotted, and the error bars represent the standard deviation. Figure S5. Southern blot analysis of the wild-type (WT) and xylose isomerase (XI) transformants. Blots were probed with sequences from A) an α-tubulin-locus area and B) ble. Restriction maps of C) the intact wild-type α-tubulin locus and D) the same locus after homologous recombination replacing the α-tubulin ORF with ble-2A-his-xi. The location of the ble and α-tubulin loci probes are indicated as black rectangles. HR represents the approximately 1kb homology arms available for homologous recombination corresponding to the α-tubulin promoter and terminator regions flanking the ble-2A-his-xi construct. Genomic DNA was digested with HindIII and ScaI. A 2.9 kb band is expected for wild type with the α-tubulin area probe. Based on this hypothetical knockout transformant map, a 5.7 kb band is expected with both probes. The molecular weight markers (MW) and corresponding sizes are indicated. Figure S6. Western blot analysis of cell extracts from wild-type T18 (WT) and XI transformants. Blots were probed with A) anti-2A antibodies and B) anti-his antibodies. Protein extracts were incubated for 30 min at 37°C or 5 min at 100°C prior separation by SDS-PAGE. Arrows indicate the bands representing Ble-2A-His-XI (1), His-XI (2), and Ble-2A (3). Figure S7. In vitro activity assays with cell extracts from wild-type and XI transformants In vitro activity assays with cell extracts from wild-type and XI transformants. Experiments were done in triplicate with A) xylose or B) xylulose at 50°C. Error bars represent standard deviation. Symbols: diamonds, wild-type; squares, XI 4; triangles, XI 6; hexagons, XI 8; and circles, XI 16. Figure S8. Southern blot analysis of XI-XK transformants. Blots were probed with sequences specific to A) an α-tubulin area, B) ble, and C) aph7. D) The restriction map of the pJB47 plasmid encoding the α-tubulin promoter-aph7-2A-xylB-α-tubulin terminator construct used to transform the parental strain XI 16. The location of aph7 probe is indicated as a black bar. HR represents the homology arms available for homologous recombination. Genomic DNA was digested with ScaI-SbfI or ScaI-HindIII. The order of the samples is the same on all blots. The expected band sizes for the WT α-tubulin loci detected by the α-tubulin probe are 7.8 kb and 2.9 kb for the ScaI-SbfI and ScaI-HindIII digests, respectively. The molecular weight markers (MW) and corresponding sizes are shown. Figure S9. Southern blot and qPCR analyses of Wild-type (WT), XI 8 and XB transformants. Blots were probed with sequences from A) an α-tubulin-locus area and B) ble. C) Restriction map of the α-tubulin locus after homologous recombination replacing the α-tubulin ORF with ble-2A-xylB. The location of the ble and α-tubulin loci probes is indicated as black rectangles. HR represents the approximately 1kb homology arms available for homologous recombination corresponding to the α-tubulin promoter and terminator regions flanking the ble-2A-xylB construct. Genomic DNA was digested with StuI or NotI. Based on this hypothetical knockout XB transformant map, the NotI digest should result in a 2.8 kb band with the α-tubulin probe and the StuI digest should result in a 2.8 kb band with the ble probe. The molecular weight markers (MW) and corresponding sizes are indicated. D) The gene copy number of the transgenic ble gene was measured by qPCR. Error bars represent the higher and lower relative quantity limits. Figure S10. Xylose isomerase and xylulose kinase activities in wild-type and XI-XK transformants. Experiments were done in triplicate with A) xylose or B) xylulose. Xylose isomerase C) and xylulose kinase D) activities were calculated from the xylulose reactions. The error bars represent standard deviations. Symbols: diamonds, WT; circles, XI 16; triangles, XB; hexagons, XI-XK 1; stars, XI-XK 3; squares, XI-XK 7.