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| − | + | Samples (about 2 mg of dried material) and 50 ��L of 2.10-3M inositol were hydrolyzed in 0.5 mL of 2 M trifluoroacetic acid for 2 h at 110��C, dried and then heated in 0.250 mL of dry 1 M HCl in methanol at 80��C for 24 h for methanolysis. After evaporation of the methanol, the sample was resuspended in 500 ��L of methanol and dried again. The methyl glycosides were then converted into their trimethylsilyl derivatives by heating the samples for 20 min at 80��C in 200 ��L of hexamethyldisilazane/trimethylchlorosilane/pyridine: 3/1/9. After evaporation of the reagent, the samples were suspended in 100 ��L of cyclohexane before being injected on a FDB-1 column (DB-1 Supelco). Chromatographic data were integrated with gas chromatography Star Workstation software (Varian), each surface being corrected according to its response factor. For comparison of amounts of cell-associated carbohydrates, total monosaccharide contents for each sample were normalized according to the OD measured at 580 nm. General DNA Procedures Restriction enzymes, T4 DNA ligase, and alkaline phosphatase were purchased from New England Biolabs (Ipswich, MA, USA) and used accordingly to the manufacturer. PCR assays were carried out with 1 ��g of P. aeruginosa chromosomal DNA, 20 pmol of each primer and failsafe Taq polymerase (Epicentre Biotechnologies, Madison, WI, USA). When necessary, PCR products and plasmids were purified with the QIAquick or QIAprep Spin Miniprep kits (QIAGEN), respectively. DNA sequencing was achieved by Genome Express (France). E. coli [commercial electrocompetent DH5�� cells (Promega, Madison, WI, USA) or S17.1 cells] and P. aeruginosa cells were transformed by electroporation or by conjugation as previously described (Bouffartigues et al., 2012). H636C Complemented Strain Construction To complement the H636 oprF mutant strain with a copy of the oprF gene into the chromosome, the mini-CTX1-araC-pBAD was constructed as follows. The region containing araC and the pBAD promoter was amplified from pJN105 (Table ?Table11) using the primer pair FaraC-pBAD and RaraC-pBAD (Table ?Table22) and cloned into pCR2.1-TA (Invitrogen, Carlsbad, CA, USA). Insert was then cut with SacI and NheI restriction enzymes and inserted into mini-CTX1 using the SacI and SpeI sites. A 1113 bp DNA fragment containing the oprF gene (PA1777) was amplified by PCR using the primer pair FoprFPstI and RoprFHindIII (Table ?Table22). The PCR product was digested with PstI and HindIII and ligated into the PstI-HindIII digested mini-CTX1-araC-pBAD vector to [https://en.wikipedia.org/wiki/ALPI ALPI] create the mini-CTX1-araC-pBAD-oprF. The sequence of this construct was verified by DNA sequencing. This vector was constructed in DH5��, purified and transferred into E. coli SM10 strain. The mini-CTX1-araC-pBAD-oprF vector was mobilized from E. coli SM10 into H636 by conjugation. Transconjugants were selected onto PIA agar plate containing 250 ��g.ml-1 of tetracyclin. | |
Версія за 11:39, 29 травня 2017
Samples (about 2 mg of dried material) and 50 ��L of 2.10-3M inositol were hydrolyzed in 0.5 mL of 2 M trifluoroacetic acid for 2 h at 110��C, dried and then heated in 0.250 mL of dry 1 M HCl in methanol at 80��C for 24 h for methanolysis. After evaporation of the methanol, the sample was resuspended in 500 ��L of methanol and dried again. The methyl glycosides were then converted into their trimethylsilyl derivatives by heating the samples for 20 min at 80��C in 200 ��L of hexamethyldisilazane/trimethylchlorosilane/pyridine: 3/1/9. After evaporation of the reagent, the samples were suspended in 100 ��L of cyclohexane before being injected on a FDB-1 column (DB-1 Supelco). Chromatographic data were integrated with gas chromatography Star Workstation software (Varian), each surface being corrected according to its response factor. For comparison of amounts of cell-associated carbohydrates, total monosaccharide contents for each sample were normalized according to the OD measured at 580 nm. General DNA Procedures Restriction enzymes, T4 DNA ligase, and alkaline phosphatase were purchased from New England Biolabs (Ipswich, MA, USA) and used accordingly to the manufacturer. PCR assays were carried out with 1 ��g of P. aeruginosa chromosomal DNA, 20 pmol of each primer and failsafe Taq polymerase (Epicentre Biotechnologies, Madison, WI, USA). When necessary, PCR products and plasmids were purified with the QIAquick or QIAprep Spin Miniprep kits (QIAGEN), respectively. DNA sequencing was achieved by Genome Express (France). E. coli [commercial electrocompetent DH5�� cells (Promega, Madison, WI, USA) or S17.1 cells] and P. aeruginosa cells were transformed by electroporation or by conjugation as previously described (Bouffartigues et al., 2012). H636C Complemented Strain Construction To complement the H636 oprF mutant strain with a copy of the oprF gene into the chromosome, the mini-CTX1-araC-pBAD was constructed as follows. The region containing araC and the pBAD promoter was amplified from pJN105 (Table ?Table11) using the primer pair FaraC-pBAD and RaraC-pBAD (Table ?Table22) and cloned into pCR2.1-TA (Invitrogen, Carlsbad, CA, USA). Insert was then cut with SacI and NheI restriction enzymes and inserted into mini-CTX1 using the SacI and SpeI sites. A 1113 bp DNA fragment containing the oprF gene (PA1777) was amplified by PCR using the primer pair FoprFPstI and RoprFHindIII (Table ?Table22). The PCR product was digested with PstI and HindIII and ligated into the PstI-HindIII digested mini-CTX1-araC-pBAD vector to ALPI create the mini-CTX1-araC-pBAD-oprF. The sequence of this construct was verified by DNA sequencing. This vector was constructed in DH5��, purified and transferred into E. coli SM10 strain. The mini-CTX1-araC-pBAD-oprF vector was mobilized from E. coli SM10 into H636 by conjugation. Transconjugants were selected onto PIA agar plate containing 250 ��g.ml-1 of tetracyclin.
