Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Four native plasmids from Z. mobilis ATCC 10988 have been used as the basis for the construction of shuttle vectors: pZMO1 (1,651 bp) [ ], pZMO2 (1,669 bp) [ - ], pZM2 (pZMO3; 2,749 bp) [ , , ] and pZM3 (pZMOB05; 4,023 bp) [ , ].

Techniques: Plasmid Preparation, Mutagenesis

Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Our results indicate that shuttle vectors containing the replicon from the pZMO7 (pZA1003) native plasmid from Z. mobilis NCIMB 11163 may be stably maintained in multi-copy levels for more than 50 generations in the ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains, in the absence of a selectable marker.

Techniques: Plasmid Preparation, Mutagenesis

Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: To determine the potential utility of pZMO7-derived shuttle vectors for heterologous gene expression in Z. mobilis , we first investigated the stability of pZ7C within three different strain lineages: NCIMB 11163, ATCC 29191 (the phenotypic centrotype strain) [ ], and CU1 Rif2 (which is derived from ATCC 10988) [ , ].

Techniques: Plasmid Preparation, Mutagenesis

Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Plasmid copy number determination for pZ7C and pZ7-184 in Z. mobilis NCIMB 11163, CU1 Rif2 and ATCC 29191 strains

Article Snippet: To determine the potential utility of pZMO7-derived shuttle vectors for heterologous gene expression in Z. mobilis , we first investigated the stability of pZ7C within three different strain lineages: NCIMB 11163, ATCC 29191 (the phenotypic centrotype strain) [ ], and CU1 Rif2 (which is derived from ATCC 10988) [ , ].

Techniques: Plasmid Preparation

Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Quantitative PCR (qPCR) analysis of pZ7C stability in Z. mobilis NCIMB 11163, CU1 Rif2 and ATCC 29191 strains cultured in media lacking chloramphenicol. The plasmid copy numbers of the pZ7 shuttle vector were monitored daily using qPCR, during iterative sub-culturing of the respective recombinant strains in RM media lacking chloramphenicol. Experiments are analogous to those shown in Figure 3. See methods section for detailed experimental procedures.

Article Snippet: To determine the potential utility of pZMO7-derived shuttle vectors for heterologous gene expression in Z. mobilis , we first investigated the stability of pZ7C within three different strain lineages: NCIMB 11163, ATCC 29191 (the phenotypic centrotype strain) [ ], and CU1 Rif2 (which is derived from ATCC 10988) [ , ].

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Plasmid Preparation, Recombinant

Journal: BMC Microbiology

Article Title: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

doi: 10.1186/1471-2180-14-68

Figure Lengend Snippet: Analysis of pZ7-GST-fusion protein expression patterns and affinity-purified protein complexes in Z. mobilis. 15% SDS-polyacrylamide gels (Coomassie Blue-stained) of proteins obtained after glutathione-affinity chromatography of cell lysates prepared from cultures of wild-type or transformant strains of Z. mobilis containing pZ7-GST, or pZ7-GST-derived expression vectors. Panel A : Z. mobilis ATCC 29191 wild type and plasmid transformed strains grown under semi-aerobic conditions. Panel B : Z. mobilis ATCC 29191 wild type and plasmid transformed strains grown under anaerobic conditions. Panel C : Z. mobilis CU1 Rif2 wild type and plasmid transformed strains grown under semi-aerobic conditions. Panel D : Z. mobilis CU1 Rif2 wild type and plasmid transformed strains grown under anaerobic conditions. The eluted protein fractions shown in lanes 1-8 are equivalent in Panels A-D. Red arrows indicate the positions of the respective pZ7C-GST-fusion proteins. Lane 1 : Benchmark protein ladder; lane 2 : wild type Z. mobilis strain (no shuttle vector); lane 3 : pZ7-GST; lane 4 : pZ7-GST-AcpP; lane 5 : pZ7-GST-KdsA; lane 6 : pZ7-GST-DnaJ; lane 7 : pZ7-GST-Hfq; lane 8 : pZ7-GST-HolC. Panel E : From left to right, identities of the proteins (co-purifying complexes) obtained from lysates of wild type (wt) Z. mobilis ATCC 29191; Z. mobilis ATCC 29191/pZ7C-GST; Z. mobilis ATCC 29191/pZ7C-GST-AcpP; and Z. mobilis ATCC 29191/pZ7C-GST-KdsA; grown under semi-aerobic conditions. ZM-GST: native glutathione S-transferase domain protein (ZZ6_0208); Glo: glyoxalase/bleomycin resistance protein/dioxygenase (ZZ6_1397); Recombinant GST: heterologous recombinant GST expressed from pZ7-GST; GST-AcpP: recombinant GST-AcpP fusion protein; GST-KdsA: recombinant GST-KdsA fusion protein; PDC: pyruvate decarboxylase (ZZ6_1397); AcpS: holo-acyl-carrier-protein synthase (ZZ6_1409); PyrG: CTP synthase (ZZ6_1034); DnaK: chaperone protein DnaK (ZZ6_0619); Tsf: translation elongation factor Ts (ZZ6_0173); Tuf: translation elongation factor Tu (ZZ6_0750); FabZ: (3R)-hydroxymyristoyl-ACP dehydratase (ZZ6_0182); G3P: glyceraldehyde-3-phosphate dehydrogenase (ZZ6_1034).

Article Snippet: To determine the potential utility of pZMO7-derived shuttle vectors for heterologous gene expression in Z. mobilis , we first investigated the stability of pZ7C within three different strain lineages: NCIMB 11163, ATCC 29191 (the phenotypic centrotype strain) [ ], and CU1 Rif2 (which is derived from ATCC 10988) [ , ].

Techniques: Expressing, Affinity Purification, Staining, Affinity Chromatography, Derivative Assay, Plasmid Preparation, Transformation Assay, Recombinant