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A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus.

Thanbichler M, Iniesta AA, Shapiro L - Nucleic Acids Res. (2007)

Bottom Line: This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)).Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter.Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. thanbichler@mpi-marburg.mpg.de

ABSTRACT
Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

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Integration Plasmids. This schematic shows different sets of vectors designed for (A) integrating constructs at a chromosomal site of interest, (B) inserting a minimal Pxyl or Pvan fragment upstream of a defined chromosomal locus, (C) creating C-terminal protein fusions encoded at the native locus of the gene of interest, and generating inducible (D) C-terminal or (E) N–terminal protein fusions encoded at the xylX or vanA locus. Plasmid names are determined as follows: each construct carries the prefix ‘p’, followed by a string of terms that describes its characteristic features and resistance gene. The individual terms are shown in red print and are to be lined up from left to right to yield the correct designation. For each category of plasmids, the name of one construct is given as an example. The different MCS (MCS, A-E) are detailed in Figure 7. The following gene names were used: RK2 origin of transfer (oriT), E. coli rrnB T1T2 transcriptional terminator (ter), pMB1 origin of replication (oriV), aminoglycoside adenylyltransferase (Spc/StrR, aadA), neomycin phosphotransferase I (KanR, nptI), rifampicin ADP-ribosyltransferase (RifR, parr-2), AAC(3)-I aminoglycoside acetyltransferase (GentR, aacC1), tetracycline efflux permease (TetR, tetA) and repressor (tetR), chloramphenicol acteyltransferase (CamR, cat), AAC(3)-IV aminoglycoside acetyltransferase (Apr/GentR, aacC4), enhanced green fluorescent protein (egfp), enhanced yellow fluorescent protein (eyfp), enhanced cyan fluorescent protein (ecfp), Venus (venus), Cerulean (cerulean), mCherry (mCherry), FLAG-tag (flag), Strep-tag II (strep), tetracysteine tag (tcys). Plasmids are shown to scale, except where indicated by breaks or dotted lines.
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Figure 6: Integration Plasmids. This schematic shows different sets of vectors designed for (A) integrating constructs at a chromosomal site of interest, (B) inserting a minimal Pxyl or Pvan fragment upstream of a defined chromosomal locus, (C) creating C-terminal protein fusions encoded at the native locus of the gene of interest, and generating inducible (D) C-terminal or (E) N–terminal protein fusions encoded at the xylX or vanA locus. Plasmid names are determined as follows: each construct carries the prefix ‘p’, followed by a string of terms that describes its characteristic features and resistance gene. The individual terms are shown in red print and are to be lined up from left to right to yield the correct designation. For each category of plasmids, the name of one construct is given as an example. The different MCS (MCS, A-E) are detailed in Figure 7. The following gene names were used: RK2 origin of transfer (oriT), E. coli rrnB T1T2 transcriptional terminator (ter), pMB1 origin of replication (oriV), aminoglycoside adenylyltransferase (Spc/StrR, aadA), neomycin phosphotransferase I (KanR, nptI), rifampicin ADP-ribosyltransferase (RifR, parr-2), AAC(3)-I aminoglycoside acetyltransferase (GentR, aacC1), tetracycline efflux permease (TetR, tetA) and repressor (tetR), chloramphenicol acteyltransferase (CamR, cat), AAC(3)-IV aminoglycoside acetyltransferase (Apr/GentR, aacC4), enhanced green fluorescent protein (egfp), enhanced yellow fluorescent protein (eyfp), enhanced cyan fluorescent protein (ecfp), Venus (venus), Cerulean (cerulean), mCherry (mCherry), FLAG-tag (flag), Strep-tag II (strep), tetracysteine tag (tcys). Plasmids are shown to scale, except where indicated by breaks or dotted lines.

Mentions: The most basic set of constructs comprises a number of general use integration vectors, which have been optimized for small size to maximize transformation yields (Figure 6A). They all have in common the RK2 origin of transfer, allowing conjugative transfer with the help of suitable host strain (18,50), the E. coli rrnB T1T2 transcriptional terminators, preventing leaky expression of cloned genes due to read-through from upstream regions (16), and a newly designed polylinker containing up to sixteen unique restriction sites (Figure 7, MCS A). Their replication is driven by the narrow host range pMB1 origin, which allows high copy numbers in E. coli but is inactive in Caulobacter. Together, these plasmids further offer a choice of seven different selective markers, thereby facilitating the generation of multiply modified strains. Five of these markers were derived from preexisting expression cassettes known to confer high-level resistance against spectinomycin, kanamycin, gentamicin, tetracycline or apramycin to Caulobacter (see Materials and Methods). In addition, novel rifampicin and chloramphenicol resistance determinants were created by combining the rickettsial parr-2 (20) and Tn9 cat genes (21), respectively, with a more efficient ribosome binding site and the R100.1 aadA promoter (51).Figure 6.


A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus.

Thanbichler M, Iniesta AA, Shapiro L - Nucleic Acids Res. (2007)

Integration Plasmids. This schematic shows different sets of vectors designed for (A) integrating constructs at a chromosomal site of interest, (B) inserting a minimal Pxyl or Pvan fragment upstream of a defined chromosomal locus, (C) creating C-terminal protein fusions encoded at the native locus of the gene of interest, and generating inducible (D) C-terminal or (E) N–terminal protein fusions encoded at the xylX or vanA locus. Plasmid names are determined as follows: each construct carries the prefix ‘p’, followed by a string of terms that describes its characteristic features and resistance gene. The individual terms are shown in red print and are to be lined up from left to right to yield the correct designation. For each category of plasmids, the name of one construct is given as an example. The different MCS (MCS, A-E) are detailed in Figure 7. The following gene names were used: RK2 origin of transfer (oriT), E. coli rrnB T1T2 transcriptional terminator (ter), pMB1 origin of replication (oriV), aminoglycoside adenylyltransferase (Spc/StrR, aadA), neomycin phosphotransferase I (KanR, nptI), rifampicin ADP-ribosyltransferase (RifR, parr-2), AAC(3)-I aminoglycoside acetyltransferase (GentR, aacC1), tetracycline efflux permease (TetR, tetA) and repressor (tetR), chloramphenicol acteyltransferase (CamR, cat), AAC(3)-IV aminoglycoside acetyltransferase (Apr/GentR, aacC4), enhanced green fluorescent protein (egfp), enhanced yellow fluorescent protein (eyfp), enhanced cyan fluorescent protein (ecfp), Venus (venus), Cerulean (cerulean), mCherry (mCherry), FLAG-tag (flag), Strep-tag II (strep), tetracysteine tag (tcys). Plasmids are shown to scale, except where indicated by breaks or dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175322&req=5

Figure 6: Integration Plasmids. This schematic shows different sets of vectors designed for (A) integrating constructs at a chromosomal site of interest, (B) inserting a minimal Pxyl or Pvan fragment upstream of a defined chromosomal locus, (C) creating C-terminal protein fusions encoded at the native locus of the gene of interest, and generating inducible (D) C-terminal or (E) N–terminal protein fusions encoded at the xylX or vanA locus. Plasmid names are determined as follows: each construct carries the prefix ‘p’, followed by a string of terms that describes its characteristic features and resistance gene. The individual terms are shown in red print and are to be lined up from left to right to yield the correct designation. For each category of plasmids, the name of one construct is given as an example. The different MCS (MCS, A-E) are detailed in Figure 7. The following gene names were used: RK2 origin of transfer (oriT), E. coli rrnB T1T2 transcriptional terminator (ter), pMB1 origin of replication (oriV), aminoglycoside adenylyltransferase (Spc/StrR, aadA), neomycin phosphotransferase I (KanR, nptI), rifampicin ADP-ribosyltransferase (RifR, parr-2), AAC(3)-I aminoglycoside acetyltransferase (GentR, aacC1), tetracycline efflux permease (TetR, tetA) and repressor (tetR), chloramphenicol acteyltransferase (CamR, cat), AAC(3)-IV aminoglycoside acetyltransferase (Apr/GentR, aacC4), enhanced green fluorescent protein (egfp), enhanced yellow fluorescent protein (eyfp), enhanced cyan fluorescent protein (ecfp), Venus (venus), Cerulean (cerulean), mCherry (mCherry), FLAG-tag (flag), Strep-tag II (strep), tetracysteine tag (tcys). Plasmids are shown to scale, except where indicated by breaks or dotted lines.
Mentions: The most basic set of constructs comprises a number of general use integration vectors, which have been optimized for small size to maximize transformation yields (Figure 6A). They all have in common the RK2 origin of transfer, allowing conjugative transfer with the help of suitable host strain (18,50), the E. coli rrnB T1T2 transcriptional terminators, preventing leaky expression of cloned genes due to read-through from upstream regions (16), and a newly designed polylinker containing up to sixteen unique restriction sites (Figure 7, MCS A). Their replication is driven by the narrow host range pMB1 origin, which allows high copy numbers in E. coli but is inactive in Caulobacter. Together, these plasmids further offer a choice of seven different selective markers, thereby facilitating the generation of multiply modified strains. Five of these markers were derived from preexisting expression cassettes known to confer high-level resistance against spectinomycin, kanamycin, gentamicin, tetracycline or apramycin to Caulobacter (see Materials and Methods). In addition, novel rifampicin and chloramphenicol resistance determinants were created by combining the rickettsial parr-2 (20) and Tn9 cat genes (21), respectively, with a more efficient ribosome binding site and the R100.1 aadA promoter (51).Figure 6.

Bottom Line: This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)).Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter.Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. thanbichler@mpi-marburg.mpg.de

ABSTRACT
Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

Show MeSH
Related in: MedlinePlus