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
Construction of GFP labeling shuttle expression vector and preparation of restitution Lactobacillus.
GFP labeling shuttle expression vector pW425et-GFP was constructed by inserting green fluorescent protein(GFP)based on erythromycin drug resistance gene /thyA gene dipl-bolting pressure shuttle expression vector pW425et.The expression stability of GFP was estimated by consecutive fluorescence intensity detection.Results of enzyme digested and PCR identify of pW425et-GFP were consistent with those expected.SDS-PAGE showed that 27 kD GFP had been expressed.Green fluorescent which could be observed in the blue light were of higher stability after 30 serial passages.Results showed that GFP labeling shuttle expression vector has been constructed successfully.It establishes the foundation for the research of field planting axiom in vivo as well as the development of the food-grade and the vaccine-grade lactic acid bacteria production of lactobacillus as a physiobacterium vector.
Shuttle vector
Aequorea victoria
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The production of green fluorescent protein (GFP) in Methylobacterium extorquens was studied by creating four different constructs using pJB3KmD, pRK310 and pVK101 vectors, as well as pLac and soluble methane monooxygenase (sMMO) promoters. Plasmids were introduced into the cells by electroporation. Expression of GFP by selected clones was evaluated by growing cells in complex or defined media. The use of pRK310 as an expression vector containing the lacZ promoter resulted in a 100-fold increase of GFP production when compared to cells containing the pLac-GFP-pJB3KmD construct. Higher production of GFP was observed also in cells containing pLac-GFP-pRK310 and pmmoX-GFP-pVK101 constructs. While the transcriptional regulation of the smmo gene in Methylosinus trichosporium OB3b is known to be copper-dependent, expression of GFP by M. extorquens clones harboring pmmoX-promoters was not strongly controlled by the presence of copper in the medium. The production of GFP was generally constant throughout the growth of M. extorquens carrying the pLac-GFP-pRK310 construct. GFP yields varied between 850 and 1000 microg of GFP g biomass(-1). However, the yield of GFP in cells carrying pmmoX-GFP-pVK101 was somewhat reduced after the mid-exponential phase of growth.
Methylobacterium
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The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.
Shuttle vector
Replicon
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The replicon of Pseudomonas plasmid DNA from pUCP19, and a new strong promoter PP303, cloned from Pseudomonas fluorescens P303 strain, were inserted into plasmid pET-29a, a high level expression vector of Escherichia coli. The recombinant shuttle expression plasmid pQMV(4.6 kb) and pGMP(5.6 kb) were obtained respectively. The vector pGMP containing gfp gene was especially available for detection and monitoring. The stability of the two expression vectors were 100%(120 h) in P303 strain of Pseudomonas fluorescens. Both recombinant vectors will be powerful for research on gene expression, regulation and construction of engineered strains of Pseudomonas.
Shuttle vector
Pseudomonas fluorescens
Replicon
Strain (injury)
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Results of development of shuttle expressing plasmid vector Escherichia coli-Lactobacillus which allowed high level expression of heterologous genes in lactobacilli are represented. Vector pTRKH2 which is able to replicate in E. coli and in wide range of Gram-positive bacteria was used as the base. In order to provide high level of cloned gene expression constitutive-active synthetic promoter, site of initiation of translation, and terminator of transcription were introduced in the vector. Functional activity of this vector was confirmed using green fluorescent protein (GFP) gene from Aequoria victoria. Transformation of model strain by gfp gene-carrying plasmid resulted in appearance of typical fluorescent phenotype.
Aequorea victoria
Shuttle vector
Heterologous
Multiple cloning site
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A modified gfp gene from Aequorea victoria, encoding a variant of the green fluorescent protein (GFP), was subcloned into the mobilizable plasmid pMV158. gfp was placed under the control of the inducible P M promoter of the Streptococcus pneumoniae gene malM, cloned in plasmid pLS70. The P M promoter is regulated by the product of the pneumococcal malR gene, which is inactivated by growing the cells in maltose-containing media. By homologous recombination, the P M–gfp construction was integrated into the host chromosome in a single copy. In both conditions (single and multiple copies), the pneumococcal cells were able to express GFP in an inducible or constitutive form, depending on whether the S. pneumoniae strain harboured a wild-type or a mutant malR gene. Quantification of the levels of GFP expressed by cultures supplemented with sucrose or maltose as carbon sources was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed pneumococcal cells expressing GFP in mixed cultures to be distinguished from those not carrying the gfp gene.
Aequorea victoria
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The gene gfpmut3a with specific promoter of Bacillus cereus were cloned into a shuttle plasmid pHT304 between E.coli and B.thuringiensis . It was then introduced into recipient strains of BMB171, CryB, IPS78-11, 4Q7 and HD80-21 by electroporation. The results showed that green fluorescence could only be detected in acrystalliferous strains.
Bacillus thuringiensis
Bacillaceae
Shuttle vector
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Marking biocontrol bacteria is an essential step to monitor bacterial behavior in natural environments before application in agricultural ecosystem. In this study, we presented the simple green fluorescent protein (GFP) reporter system driven by the promoter active in Bacillus species for tagging of the biocontrol bacteria. A constitutive promoter P43 from Bacillus subtilis was fused to an enhanced promoterless gfp gene by overlap extension PCR. The GFP expression was demonstrated by the high fluorescence intensity detected in B. subtilis and Escherichia coli transformed with the P43-gfp fusion construct, respectively. The GFP reporter system was further investigated in two bacterial biocontrol strains B. licheniformis and Pseudomonas fluorescens. When the reconstructed plasmid pWH34G was introduced into B. licheniformis, GFP level measured with the fluorescence intensity in B. licheniformis was almost equivalent to that in B. subtilis. However, GFP expression level was extremely low in other biocontrol bacteria P. fluorescens by transposon based stable insertion of the P43-gfp construct into the bacterial chromosome. This study provides information regarding to the efficient biomarker P43-gfp fusion construct for bio-control Bacillus species.
Pseudomonas fluorescens
Bacillus licheniformis
Bacillus thuringiensis
mCherry
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Pseudomonas stutzeri
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The gfp sequence was amplified from pAD123 by PCR.pAXc-gfp was constructed by ligation of the gfp PCR product and pAXc digested by BamHI.pAXc-gfp was linearized by Pst I and transformed into B.subtilis B411.The linearized plasmid should be integrated at the lacA locus on B.subtilis B411 chromosome DNA.The recombinants were screened by chloramphenicol resistance(5 μg/mL) phenotype.Recombinant named B412 could express GFP stably without antibiotic pressure.
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