Expression of Green Fluorescent Protein Gene in Gluconacetobacter xylinus
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Green fluorescent protein(GFP)as a reporter protein is widely used in signal conduction and gene expression and regulation in microbiology. In this research,GFP genes were successfully expressed in Gluconacetobacter xylinus with shuttle plasmid p MV24. GFP genes were cloned from p MUTIN-gfp+ plasmid and connected to p MV24 to build prokaryotic expression plasmid p MV24-gfp+. Electrotransformation was applied to introduce p MV24-gfp+ into G. xylinus. The transformant exhibits bright green fluorescence when exposed to blue light in the ultraviolet range of fluorescence microscope. It can provide very important theoretical basis for the observation of cell movement and the analysis of kinesin of G. xylinus under chemotaxis.Cite
The anti-nematode(Alcaligenes faecalis)strain BC2000 was chromosomally tagged with the marker genes gfp and luxAB by electroporation with a pUTmini-Tn5 tmnsposon vector containing the gfp/luxAB dual markers.Transformants were screened for the green fluorescence phenotype of the gfp marker by fluorescence stereomicroscopy and one tagged strain was selected for further experiments and designated as strain BC2001.A 700 bp fragment of the gfp gene was amplified from the BC2001 genome DNA using two gfp specific primers.Morphologically the cells of the engineered sWain were similar to the original sWain as determined by laser scanning confocal microscopy.Luciferase activity of BC2001 increased rapidly during log-phase growth in LB medium,but decreased in stationary phase.These results confirmed that the dual marker was insetted successfully into the BC2000 genome through electroporation,and highly expressed in transformant BC2001 under the control ofpsbA promoter.
Alcaligenes faecalis
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The PSJ2 promoter cloned from Bacillus subtilis was spliced to gfp gene by overlap extension PCR.Thus the fused translational expression cassette PSJ2-gfp was constructed.After being digested by RcoR I and Pst I,PSJ2-gfp expression cassette was inserted into Bacillus subtilis vector pUS186 to give pUS186PGFP,which was then transformed into Bacillus amyloliquefaciens strain TB2 to give strain TB2GFP.The strain TB2GFP appeared weak green under blue light.The inhibiting effect of this strain on the growth of cucumerium,Fusarium oxyporum f.sp.,was not remarkably different from that of TB2.
Bacillus amyloliquefaciens
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Expression cassette
Bacillus (shape)
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Promoter 4412 of plasmid pGFP4412 was replaced by rpsD promoter of Bacillus subtilis strain 168 to obtain a new vector pS4GFP in which the gfp gene could strongly express under the control of rpsD promoter. Plasmid pS4GFP was transformed into the endophytic B. subtilis strain BS-2, and a fine gfp-labeled strain BS-2-gfp was obtained. The tests showed the strain could keep promoting plant growth and inhibiting plant pathogens. The strain also could appear bright green under blue light and the recombined plasmid was stable in the bacterial cells. The colonizing inside of cabbages with the strain showed that the bacteria could adhere to the surface of roots, and could be colonized in roots, stems and leaves. After inoculation for 50 days, the labeled bacteria could still be isolated from the cabbages. Fig 5, Ref 17
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The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/R and primers gfpF/R, which were designed according to the gfp gene and promoter sequence of xylase operon from Bacillus subtilis 168, and the DNA template plasmids pHT315-xyIR and pGFPuv. Furthermore, the fused translational expression cassette PxylR-gfp was constructed using overlapping PCR technique with the primers pair PxyF/gfpR and the mixture of above PCR production. After being digested by Kpn I and Sph I , PxylR-gfp expression cassette was inserted into E. coli-B. thuringiensis shuttle vecter pHT315 and E. coli-B. subtilis shuttle vecter pRP22, and the resulted recombinant plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis lab strain 168 and the resulted transformants are bright green performance under 365 nm UV light. However, only pGFP22 can be introduced into the natural strain B916. The transformants containing pGFP22 have bright green performance under 365 nm UV light and was named B916-gfp. Antifungal activities testing results proved that there is no obvious difference between B916 and the engineered strains B916-gfp. Research results also showed that the stability of B916-gfp was 94% after growth about 175 generations at 37 degrees C, and the losing rate of plasmid was less than 3.5 x 10(-4) per generation.
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Bacillus thuringiensis
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Cereus
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We employed Pvgb,an oxygen-dependent promoter of VHb,for self-tuning regulation of green fluorescent protein(GFP) expression according to the natural transition of dissolved oxygen(DO) level during culture.A series of plasmid vectors were created using a promoter of varying size(Pvgb-L or Pvgb-S) to drive expression of GFP.Transcription and protein accumulation were compared between each expression vector.Pvgb transcript level appears to parallel GFP protein accumulation.GFP expression in the Escherichia coli strain was very largely affected by variation of aeration environment.Strong expression of xylose genes were also observed using low aeration in recombinant E.coli W3110(ΔpflB,ΔadhE) harboring pSK(Pvgb,xylA,xylB,talA,tktA).These vectors induced by Pvgb should be useful for overexpression of heterologous proteins and potential metabolic engineering of E.coli strains.
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A 138-bp EcoO109/HinfI fragment of Streptococcus thermophilus plasmid pER341 (2798 bp) including the promoter sequence of the heat stress protein gene hsp16.4 was tested in vector constructs for ability to activate the promoterless green fluorescent protein gene (gfp) from a jelly fish in Escherichia coli, S. thermophilus, and lactococci. ST(Phsp) promoted gfp expression in transformed hosts as evidenced by the presence of green fluorescent (GFP(+)) colonies under UV illumination. The results confirmed the potential of ST(Phsp) as a functional promoter in heterologous gene expression in dairy fermentation bacteria.
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Heterologous expression
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UNLABELLED OBJECTIVE; We cloned the promoter of glycerol-3-phosphate dehydrogenase gene(CgGPD) from the Candida glycerinogenes, and studied its functional regulation under high osmotic stress condition. METHODS We amplified the 950 bp promoter of CgGPD from C. glycerinogenes and the green fluorescent protein gene (gfp) from pCAMBIA1302 vector by PCR and introduced them into a modified vector pYX212-zeocin simultaneously. The recombinant plasmid pYX212-zeocin harboring both the promoter of CgGPD and gene gfp was transformed into S. cerevisiae W303-1A by electroporation. In the medium containing glucose with different concentrations for culturing the recombinant strain S. cerevisiae W303-1A-GFP the green fluorescence was detected by fluorescent microscopy. RESULTS The gene gfp was functionally expressed under the control of the promoter of CgGPD in S. cerevisiae. Furthermore, the expression of the gene gfp at different level was conducted by the different osmotic stress for the recombinant strain. The green fluorescence was less intensive when the concentration of glucose was low for culturing the recombinant strain, but it became much more intensive when the concentration of glucose increased. CONCLUSION The promoter of CgGPD is an inducible promoter that can be induced significantly by the high concentration of glucose. The promoter will facilitate further studies on the mechanism of glycerol synthesis from C. glycerinogenes WL2002-5 under osmotic stress conditions.
Osmotic shock
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