All labs need to do PCR set-up in a different room from the site where PCR is done or PCR products are handled. I have heard of proven cases where PCR product from a lab on a different floor of the building contaminated a lab, carried in by a coworkers contaminated glove or something. Although every cell in the organism contains a few mitochondria, the PCR pruct produced by PCR ends up many millions of fold more concentrated than any sample DNA (the molar concentration of the product is enormous even if the micrograms of DNA per volume are low). The very best labs can nearly eliminate it, but all labs need positive and negative controls constantly run to ensure that if any contamination happens it is caught early before any efforts are wasted analyzing contaminated results. PCR contamination happens to everyone, it does not indicate "sloppy work". Glycosyl-residue compositions of microbe pellets.If you gene in your organism should be "pure", then the mix indicates a problem, most likely PCR contamination. * indicates unidentified residue, but not a sugar. The region of the total ion count for xylose- (Xyl) and apiose- (Api) alditol-acetate derivatives is expanded. Standard (Std) contains authentic xylose and apiose. coli engineered to overexpress BtbUGDH + SpUAS with X. GC-MS analysis of alditol-acetate derivatives from cell pellet fractions of E. (B) Average of all ions at 11.0 min (MS, top panel) and average of all fragmented ions at 11.0 min (MS/MS, bottom panel). (A) Total ion count (TIC, dashed line) and XIC - ions diagnostic for UDP-pentose ( m/z 535.0, solid line). HPLC-purified in vitro synthesized UDP-apiose. Data is the average of three independent samples and displayed with calculated standard error (SE) bars. Relative luminescence is fold signal over background measured by subtracting blank (enzyme without UDP-sugar) and dividing by background (UDP-sugar with boiled enzyme). Equal volume of UDP-Glo detection reagent added to stop reaction for 1 hr. Assays of 50 mM potassium phosphate pH 7.5, 0.1 mM UDP-sugar, 0.5 μg enzyme in 10 μl total volume reacted at room temperature for 1–1.5 hr. (B) Bar graph of relative luminescence from UDP-Glo assays. coli cells induced to express XpXylT, XpApiT, and empty vector control with expected sizes of XpXylT and XpApiT: 32.9 and 41.4 kDa, respectively. coli.Īctivity of purified XpXylT and XpApiT proteins. Park’s nucleotide is a UDP-MurNAc-pentapeptide that is used as an internal standard for nucleotide-sugar detection as it is abundantly made in E. Extracted ion chromatograms (XICs) of - ions diagnostic for UDP-pentose ( m/z 535.0, solid line), UDP-hexuronic acid ( m/z 579.0, dashed line) and Park’s nucleotide ( m/z 595.6, dotted line) are displayed. coli cells induced to express genes encoding BtbUGDH and SpUAS only BtbUGDH and SpUAS along with a putative N-acetyl-glucosamine transferase from Bacillus thuringiensis (BtGlyT046), XpXylT, XpApiT, or XpApiT with XpXylT or only XpApiT with XpXylT as control (bottom panel). (A) top panel elution of standard (Std): UDP-GlcA and UDP-Xyl Nucleotide sugars were extracted from E. ![]() In microbe activity of XpXylT and XpApiT.Īnalysis of in microbe nucleotide sugars by HILIC-LC-ESI-MS/MS. For full amino acid sequence alignment with organism and gene names see S1 and S2 Figs. Distance scale represents difference between sequences in substitutions per site. Alignments were made using Clustal Omega and the trees generated using Dendroscope. ![]() Amino acid sequences from representatives of pfam05686 and pfam13704 selected by the CDD were used. (B) phylogenetic analysis of XpApiT (PPU69163) and XpXylT (PPU69164). Operon schema was generated using SnapGene Viewer version 3.1.4 (GSL Biotech, Chicago, IL). 35 and -10 promoters were predicted using Bacterial Promoter Prediction website (BacPP, ). (A) Gene organization of the apiose operon in X.
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