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Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, Stolz T, Dersch P - PLoS ONE (2011)

Bottom Line: The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis.The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs.A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany.

ABSTRACT
A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

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The pFU series of fusion vectors.(A) Composition of the fusion vectors of the pFU series. The vectors are made up of three interchangeable modules: (i) a resistance cassette with one of four different resistance genes encoded on a AatII/SacI fragment, (ii) a SacI/AvrII fragment harboring one of five different origins of replication, and (iii) a XhoI/NotI fragment encoding the reporter gene with or without ribosome binding site downstream of a multiple cloning site containing seven unique restriction sites. +: SpeI site is not unique in plasmids carrying the pSC101* origin, (B) Representative collection of the pFU plasmids carrying the high-copy origin ColE1, the midi-copy origins p15A and p29807, the low-copy pSC101* origin, and the suicide vector R6K combined with a mobilizable origin (mob) for conjugation. They contain ampicillin, chloramphenicol, kanamycin, or tetracycline resistance genes including their promoter and ribosome binding sites which are oriented in the opposite direction of the reporter genes luxCDABE, phoA, lacZ, gfpmut3.1, gfpmut3.1-LVA, amCyan or dsRed2 with or without their ribosome binding site (rbs).
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pone-0020425-g001: The pFU series of fusion vectors.(A) Composition of the fusion vectors of the pFU series. The vectors are made up of three interchangeable modules: (i) a resistance cassette with one of four different resistance genes encoded on a AatII/SacI fragment, (ii) a SacI/AvrII fragment harboring one of five different origins of replication, and (iii) a XhoI/NotI fragment encoding the reporter gene with or without ribosome binding site downstream of a multiple cloning site containing seven unique restriction sites. +: SpeI site is not unique in plasmids carrying the pSC101* origin, (B) Representative collection of the pFU plasmids carrying the high-copy origin ColE1, the midi-copy origins p15A and p29807, the low-copy pSC101* origin, and the suicide vector R6K combined with a mobilizable origin (mob) for conjugation. They contain ampicillin, chloramphenicol, kanamycin, or tetracycline resistance genes including their promoter and ribosome binding sites which are oriented in the opposite direction of the reporter genes luxCDABE, phoA, lacZ, gfpmut3.1, gfpmut3.1-LVA, amCyan or dsRed2 with or without their ribosome binding site (rbs).

Mentions: The basic cloning vector of the pFU series (Figure 1) was derived from vector pZE12-luc [21]. A fragment which contains the gfpmut3.1 gene from pGFPmut3.1 (Clontech) was amplified by PCR with primers I916 and I917 including a new multiple cloning site (MCS) and inserted into the XhoI/XbaI sites of pZE12-luc to generate pFU31. The dsRed2, amCyan, luxCDABE, lacZ, and phoA reporter genes were PCR-amplified from different vectors (pDsRed2, pAmCyan, pUTmini-Tn5luxCDABEKm2, pHT124, pGP20) or genomic DNA derived from E. coli DH10beta(phoA) by using the primers listed in Figure S2.


Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, Stolz T, Dersch P - PLoS ONE (2011)

The pFU series of fusion vectors.(A) Composition of the fusion vectors of the pFU series. The vectors are made up of three interchangeable modules: (i) a resistance cassette with one of four different resistance genes encoded on a AatII/SacI fragment, (ii) a SacI/AvrII fragment harboring one of five different origins of replication, and (iii) a XhoI/NotI fragment encoding the reporter gene with or without ribosome binding site downstream of a multiple cloning site containing seven unique restriction sites. +: SpeI site is not unique in plasmids carrying the pSC101* origin, (B) Representative collection of the pFU plasmids carrying the high-copy origin ColE1, the midi-copy origins p15A and p29807, the low-copy pSC101* origin, and the suicide vector R6K combined with a mobilizable origin (mob) for conjugation. They contain ampicillin, chloramphenicol, kanamycin, or tetracycline resistance genes including their promoter and ribosome binding sites which are oriented in the opposite direction of the reporter genes luxCDABE, phoA, lacZ, gfpmut3.1, gfpmut3.1-LVA, amCyan or dsRed2 with or without their ribosome binding site (rbs).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108616&req=5

pone-0020425-g001: The pFU series of fusion vectors.(A) Composition of the fusion vectors of the pFU series. The vectors are made up of three interchangeable modules: (i) a resistance cassette with one of four different resistance genes encoded on a AatII/SacI fragment, (ii) a SacI/AvrII fragment harboring one of five different origins of replication, and (iii) a XhoI/NotI fragment encoding the reporter gene with or without ribosome binding site downstream of a multiple cloning site containing seven unique restriction sites. +: SpeI site is not unique in plasmids carrying the pSC101* origin, (B) Representative collection of the pFU plasmids carrying the high-copy origin ColE1, the midi-copy origins p15A and p29807, the low-copy pSC101* origin, and the suicide vector R6K combined with a mobilizable origin (mob) for conjugation. They contain ampicillin, chloramphenicol, kanamycin, or tetracycline resistance genes including their promoter and ribosome binding sites which are oriented in the opposite direction of the reporter genes luxCDABE, phoA, lacZ, gfpmut3.1, gfpmut3.1-LVA, amCyan or dsRed2 with or without their ribosome binding site (rbs).
Mentions: The basic cloning vector of the pFU series (Figure 1) was derived from vector pZE12-luc [21]. A fragment which contains the gfpmut3.1 gene from pGFPmut3.1 (Clontech) was amplified by PCR with primers I916 and I917 including a new multiple cloning site (MCS) and inserted into the XhoI/XbaI sites of pZE12-luc to generate pFU31. The dsRed2, amCyan, luxCDABE, lacZ, and phoA reporter genes were PCR-amplified from different vectors (pDsRed2, pAmCyan, pUTmini-Tn5luxCDABEKm2, pHT124, pGP20) or genomic DNA derived from E. coli DH10beta(phoA) by using the primers listed in Figure S2.

Bottom Line: The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis.The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs.A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany.

ABSTRACT
A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

Show MeSH
Related in: MedlinePlus