Ructs that still carry the first 10 aminoacids of Wze (P.0.05). A significant reduction of the 23388095 fluorescent signal was observed in strains BCSJC004, BCSJC005 and BCSJC010 (*, P,0.01). (Bottom panel) A schematic representation of each Wze constructs is shown. White box represent the Citrine Hexokinase II Inhibitor II, 3-BP site protein while gray boxes represent different regions of Wze protein (lighter grey ?aminoacids 1 to 10, light grey ?remaining N-terminus region between aminoacids 11 to 50, dark grew ?central region between aminoacids 51?76, black box ?C-terminus region between aminoacids 178?27. Strain name are indicated below. doi:10.1371/journal.pone.0055049.g37uC, without aeration, or in tryptic soy agar (TSA, Difco) plates supplemented with 5 sheep blood (Probiologica). Tetracycline was added to the media at 1 mg/ml final concentration.DNA manipulation proceduresS. pneumoniae competent cells preparation and transformation was executed as previously described [25]. PCR products and plasmid DNA were purified with kits WizardH SV Gel and PCR Clean-up System and WizardH Plus SV Minipreps, respectively (Promega).Figure 5. Increased fluorescence resulting from the presence of the “i-tag” is due to increased protein levels. (A) mRNA encoding Citrine was quantified by Real-time PCR in strains expressing specific aminoacid sequences from Wze fused to Citrine, relatively to the mRNA for the tetracycline resistance protein which is encoded in the plasmid backbone. Strains analyzed were BCSMH031 (transformed with an empty vector), BCSMH007 (expressing full Wze-Citrine), BCSMH033 (expressing Citrine), BCSJC003 (expressing Wze (51?27) -Citrine), BCSJC004 (expressing Wze(1?0, 178?27)-Citrine), BCSJC005 (expressing Wze(1?77)-Citrine), BCSJC001 (expressing Wze(1?0)-Citrine), BCSJC007 (expressing Wze(11?0)-Citrine) and BCSJC002 (expressing Wze(1?0)Citrine). Cell extracts from these strains were separated by SDS-Page and analyzed using a Fluorescent Image Analyzer (B) and by Westernblot analysis using an antibody that recognizes Citrine protein (C), showing that fluorescence in strains containing the i-tag is due to increased protein levels and not to increased mRNA levels. doi:10.1371/journal.pone.0055049.gPCR fragments were amplified with Phusion High-fidelity DNA polymerase (Finnzymes). Restriction enzymes were from New England Biolabs. Primers used in this study are listed in Table S2.Expression of 15857111 Fluorescent Proteins in S.pneumoniaeFigure 6. The nucleotide sequence of the i-tag determines the expression of fluorescence. (Left panel) 298690-60-5 web Median fluorescence, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) of the fluorescence emitted by Citrine from the following strains: BCSMH031 (empty plasmid), BCSMH033 (expressing Citrine), BCSJC001 (expressing Wze(1?0)-Citrine), BCSJC006 (expressing Wze(1?0)*-Citrine in which the sequence encoding for Leucine was changed from TTA to CTC). At least 100 cells of each strain were quantified. (Right panel) Sequence encoding aminoacids 3? of Wze present in Wze(1?0)-Citrine and Wze(1?0)*-Citrine. doi:10.1371/journal.pone.0055049.gConstruction of plasmids for protein expression in S. pneumoniaeFor expression of CFP and GFP, not fused to any protein, we constructed plasmids pBCSMH018 and pBCSMH020 by amplification of the CFP and GFP(P5) coding sequences from plasmids pMUTIN-CFP and pTrc99A-GFP, respectively, with primers 1/2 and primers 7/8, followed by restriction and ligation to the vector backbone o.Ructs that still carry the first 10 aminoacids of Wze (P.0.05). A significant reduction of the 23388095 fluorescent signal was observed in strains BCSJC004, BCSJC005 and BCSJC010 (*, P,0.01). (Bottom panel) A schematic representation of each Wze constructs is shown. White box represent the Citrine protein while gray boxes represent different regions of Wze protein (lighter grey ?aminoacids 1 to 10, light grey ?remaining N-terminus region between aminoacids 11 to 50, dark grew ?central region between aminoacids 51?76, black box ?C-terminus region between aminoacids 178?27. Strain name are indicated below. doi:10.1371/journal.pone.0055049.g37uC, without aeration, or in tryptic soy agar (TSA, Difco) plates supplemented with 5 sheep blood (Probiologica). Tetracycline was added to the media at 1 mg/ml final concentration.DNA manipulation proceduresS. pneumoniae competent cells preparation and transformation was executed as previously described [25]. PCR products and plasmid DNA were purified with kits WizardH SV Gel and PCR Clean-up System and WizardH Plus SV Minipreps, respectively (Promega).Figure 5. Increased fluorescence resulting from the presence of the “i-tag” is due to increased protein levels. (A) mRNA encoding Citrine was quantified by Real-time PCR in strains expressing specific aminoacid sequences from Wze fused to Citrine, relatively to the mRNA for the tetracycline resistance protein which is encoded in the plasmid backbone. Strains analyzed were BCSMH031 (transformed with an empty vector), BCSMH007 (expressing full Wze-Citrine), BCSMH033 (expressing Citrine), BCSJC003 (expressing Wze (51?27) -Citrine), BCSJC004 (expressing Wze(1?0, 178?27)-Citrine), BCSJC005 (expressing Wze(1?77)-Citrine), BCSJC001 (expressing Wze(1?0)-Citrine), BCSJC007 (expressing Wze(11?0)-Citrine) and BCSJC002 (expressing Wze(1?0)Citrine). Cell extracts from these strains were separated by SDS-Page and analyzed using a Fluorescent Image Analyzer (B) and by Westernblot analysis using an antibody that recognizes Citrine protein (C), showing that fluorescence in strains containing the i-tag is due to increased protein levels and not to increased mRNA levels. doi:10.1371/journal.pone.0055049.gPCR fragments were amplified with Phusion High-fidelity DNA polymerase (Finnzymes). Restriction enzymes were from New England Biolabs. Primers used in this study are listed in Table S2.Expression of 15857111 Fluorescent Proteins in S.pneumoniaeFigure 6. The nucleotide sequence of the i-tag determines the expression of fluorescence. (Left panel) Median fluorescence, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) of the fluorescence emitted by Citrine from the following strains: BCSMH031 (empty plasmid), BCSMH033 (expressing Citrine), BCSJC001 (expressing Wze(1?0)-Citrine), BCSJC006 (expressing Wze(1?0)*-Citrine in which the sequence encoding for Leucine was changed from TTA to CTC). At least 100 cells of each strain were quantified. (Right panel) Sequence encoding aminoacids 3? of Wze present in Wze(1?0)-Citrine and Wze(1?0)*-Citrine. doi:10.1371/journal.pone.0055049.gConstruction of plasmids for protein expression in S. pneumoniaeFor expression of CFP and GFP, not fused to any protein, we constructed plasmids pBCSMH018 and pBCSMH020 by amplification of the CFP and GFP(P5) coding sequences from plasmids pMUTIN-CFP and pTrc99A-GFP, respectively, with primers 1/2 and primers 7/8, followed by restriction and ligation to the vector backbone o.
