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A new recombineering system for Photorhabdus and Xenorhabdus.

Yin J, Zhu H, Xia L, Ding X, Hoffmann T, Hoffmann M, Bian X, Müller R, Fu J, Stewart AF, Zhang Y - Nucleic Acids Res. (2014)

Bottom Line: The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter.After tetracycline induction, novel secondary metabolites were identified.Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.

View Article: PubMed Central - PubMed

Affiliation: Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.

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Recombineering with different protein combinations in E. coli and P. luminescens. (A) Results from a linear plus linear homologous recombination (LLHR) assay in E. coli (23) upon expression of Redγ (g); Plu2935 and Redγ (35-g), Plu2935/Plu2936 and Redγ(35–36-g), Plu2935/Plu2936/Plu2937 and Redγ (35-36-37-g), Plu2934/Plu2935/Plu2936 and Redγ (34-35-36-g), Plu2934/Plu2935/Plu2936/Plu2937 and Redγ (34-35-36-37-g) and full length RecE, RecT and Redγ (ETg). Expression was driven by the pBAD promoter in the pSC101-amp plasmid. (B) As in (A) except using a linear plus circular homologous recombination (LCHR) assay (23) and Redγ, Redβ and Redα (gba) was used as a reference instead of ETg. (C) Diagram of the LCHR assay used in P. luminescens. A PCR product carrying the kanamycin resistance gene (kan) flanked by 50-bp homology arms (represented by the thick lines) was integrated into the expression plasmid in place of the ampicillin resistance gene. (D) Results from the LCHR assay depicted in (C) upon expression of various protein combinations as defined in (A) and (B). Colonies were selected on kanamycin plates and counted. Error bars, SD; n = 3.
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Figure 2: Recombineering with different protein combinations in E. coli and P. luminescens. (A) Results from a linear plus linear homologous recombination (LLHR) assay in E. coli (23) upon expression of Redγ (g); Plu2935 and Redγ (35-g), Plu2935/Plu2936 and Redγ(35–36-g), Plu2935/Plu2936/Plu2937 and Redγ (35-36-37-g), Plu2934/Plu2935/Plu2936 and Redγ (34-35-36-g), Plu2934/Plu2935/Plu2936/Plu2937 and Redγ (34-35-36-37-g) and full length RecE, RecT and Redγ (ETg). Expression was driven by the pBAD promoter in the pSC101-amp plasmid. (B) As in (A) except using a linear plus circular homologous recombination (LCHR) assay (23) and Redγ, Redβ and Redα (gba) was used as a reference instead of ETg. (C) Diagram of the LCHR assay used in P. luminescens. A PCR product carrying the kanamycin resistance gene (kan) flanked by 50-bp homology arms (represented by the thick lines) was integrated into the expression plasmid in place of the ampicillin resistance gene. (D) Results from the LCHR assay depicted in (C) upon expression of various protein combinations as defined in (A) and (B). Colonies were selected on kanamycin plates and counted. Error bars, SD; n = 3.

Mentions: Various expression plasmids were electroporated into E. coli, P. luminescens and X. stockiae. The E. coli electrocompetent cells were prepared according to our established protocol (41). For P. luminescens and X. stockiae, overnight cultures containing the expression plasmids were diluted into 1.3-ml LB medium with appropriate antibiotics. The starting OD600 value was around 0.15. The fresh culture was grown at 30°C, 950 rpm for 4.5 h until the OD600 was around 0.85. After addition of the inducer L-(+)-arabinose to a final concentration of 2.5 mg/ml, the cells were grown at 30°C, 950 rpm for 30 min until the OD600 was around 1.15. Cells were then centrifuged for 30 s at 9500 rpm at 2°C. The supernatant was discarded, and the cell pellet was resuspended in 1-ml ice-cold GH buffer (10% glycerol, 2-mM HEPES, PH = 6.5) and centrifuged. The ice-cold GH buffer washing was repeated once more. After that, cells were resuspended in 30-μl ice-cold GH buffer and DNA was added. Two hundred nanogram PCR products were used in experiments of Figures 2D, 3C and 4C and E. One microgram of the PCR products was used for genome modification of P. luminescens and X. stockiae, to insert the inducible promoter in front of the gene clusters (Figure 5). Electroporation was performed using ice-cold cuvettes and an Eppendorf 2510 electroporator set at 1200 V. One milliliter LB medium was added after electroporation. The cells were incubated at 30°C for 100 min with shaking then spread on appropriate antibiotic plates.


A new recombineering system for Photorhabdus and Xenorhabdus.

Yin J, Zhu H, Xia L, Ding X, Hoffmann T, Hoffmann M, Bian X, Müller R, Fu J, Stewart AF, Zhang Y - Nucleic Acids Res. (2014)

Recombineering with different protein combinations in E. coli and P. luminescens. (A) Results from a linear plus linear homologous recombination (LLHR) assay in E. coli (23) upon expression of Redγ (g); Plu2935 and Redγ (35-g), Plu2935/Plu2936 and Redγ(35–36-g), Plu2935/Plu2936/Plu2937 and Redγ (35-36-37-g), Plu2934/Plu2935/Plu2936 and Redγ (34-35-36-g), Plu2934/Plu2935/Plu2936/Plu2937 and Redγ (34-35-36-37-g) and full length RecE, RecT and Redγ (ETg). Expression was driven by the pBAD promoter in the pSC101-amp plasmid. (B) As in (A) except using a linear plus circular homologous recombination (LCHR) assay (23) and Redγ, Redβ and Redα (gba) was used as a reference instead of ETg. (C) Diagram of the LCHR assay used in P. luminescens. A PCR product carrying the kanamycin resistance gene (kan) flanked by 50-bp homology arms (represented by the thick lines) was integrated into the expression plasmid in place of the ampicillin resistance gene. (D) Results from the LCHR assay depicted in (C) upon expression of various protein combinations as defined in (A) and (B). Colonies were selected on kanamycin plates and counted. Error bars, SD; n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4381043&req=5

Figure 2: Recombineering with different protein combinations in E. coli and P. luminescens. (A) Results from a linear plus linear homologous recombination (LLHR) assay in E. coli (23) upon expression of Redγ (g); Plu2935 and Redγ (35-g), Plu2935/Plu2936 and Redγ(35–36-g), Plu2935/Plu2936/Plu2937 and Redγ (35-36-37-g), Plu2934/Plu2935/Plu2936 and Redγ (34-35-36-g), Plu2934/Plu2935/Plu2936/Plu2937 and Redγ (34-35-36-37-g) and full length RecE, RecT and Redγ (ETg). Expression was driven by the pBAD promoter in the pSC101-amp plasmid. (B) As in (A) except using a linear plus circular homologous recombination (LCHR) assay (23) and Redγ, Redβ and Redα (gba) was used as a reference instead of ETg. (C) Diagram of the LCHR assay used in P. luminescens. A PCR product carrying the kanamycin resistance gene (kan) flanked by 50-bp homology arms (represented by the thick lines) was integrated into the expression plasmid in place of the ampicillin resistance gene. (D) Results from the LCHR assay depicted in (C) upon expression of various protein combinations as defined in (A) and (B). Colonies were selected on kanamycin plates and counted. Error bars, SD; n = 3.
Mentions: Various expression plasmids were electroporated into E. coli, P. luminescens and X. stockiae. The E. coli electrocompetent cells were prepared according to our established protocol (41). For P. luminescens and X. stockiae, overnight cultures containing the expression plasmids were diluted into 1.3-ml LB medium with appropriate antibiotics. The starting OD600 value was around 0.15. The fresh culture was grown at 30°C, 950 rpm for 4.5 h until the OD600 was around 0.85. After addition of the inducer L-(+)-arabinose to a final concentration of 2.5 mg/ml, the cells were grown at 30°C, 950 rpm for 30 min until the OD600 was around 1.15. Cells were then centrifuged for 30 s at 9500 rpm at 2°C. The supernatant was discarded, and the cell pellet was resuspended in 1-ml ice-cold GH buffer (10% glycerol, 2-mM HEPES, PH = 6.5) and centrifuged. The ice-cold GH buffer washing was repeated once more. After that, cells were resuspended in 30-μl ice-cold GH buffer and DNA was added. Two hundred nanogram PCR products were used in experiments of Figures 2D, 3C and 4C and E. One microgram of the PCR products was used for genome modification of P. luminescens and X. stockiae, to insert the inducible promoter in front of the gene clusters (Figure 5). Electroporation was performed using ice-cold cuvettes and an Eppendorf 2510 electroporator set at 1200 V. One milliliter LB medium was added after electroporation. The cells were incubated at 30°C for 100 min with shaking then spread on appropriate antibiotic plates.

Bottom Line: The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter.After tetracycline induction, novel secondary metabolites were identified.Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.

View Article: PubMed Central - PubMed

Affiliation: Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.

Show MeSH
Related in: MedlinePlus