The fluorescence intensity was normalized by dividing the fluorescence intensity at the emission wavelength nm by the OD value of the same sample. The background value was obtained from the control assays with uninduced cell suspensions. The precultured recombinant E.
The LB agar was supplemented with 0. First, we investigated the response profiles of recombinant E. After bacterial cells in logarithmic growth were exposed to 0—1.
The results are shown in Figure 3A. However, even when the concentration of IPTG reached as low as 0. The preferred concentration of IPTG was finally determined to be 0. Figure 3. Recombinant E. The optical density at nm and the mCherry fluorescence intensity were all normalized by the OD value of the induced culture. The results are shown in Figure 3B. Cultures were sampled at consecutive time intervals after IPTG exposure. It is well known that the expression of recombinant protein is determined by host genetic background, plasmid copy number, inducer concentration, culture conditions, and so on Hortsch and Weuster-Botz, ; Mahalik et al.
Thus, 0. After recombinant E. Figure 4. The optical density at nm and mCherry fluorescence intensity were all normalized by the OD value of the induced culture.
The results are shown in Figure 4B. The results are shown in Figure 5. Figure 5. Assay of the pbr promoter activities in response to lead II. This result was basically consistent with previous findings Bereza-Malcolm et al. However, no significantly higher response from the mCherry reporter system was observed with the increase of Pb II in the study. The activity of a target promoter in response to an inducer can be quantified in the liquid media.
Figure 6. Expression of the three reporters in recombinant E. Extra substrate, 0. Enzyme fragment complementation has been used as a selection marker for the rapid detection of recombinant bacteria, protein-protein interaction, high throughput screenings, and so on. The resultant stable heteromeric enzyme complex can be easily detected using a chromogenic substrate, X-gal, which forms an intense blue precipitate when hydrolyzed.
A lac promoter was first chosen as target promoter. A moderately strong ara promoter was then chosen to be the detection promoter. A weak pbr promoter was finally chosen to be the detection promoter. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. C-YH designed the experimental protocol and drafted the manuscript.
LL analyzed the data and corrected the manuscript. All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species.
Belinda Rowland , Belinda Rowland. Oxford Academic. Anjan Purkayastha. Catherine Monserrat. Yveth Casart. Howard Takiff. Kathleen A. Select Format Select format. Permissions Icon Permissions. Open in new tab Download slide. The application of luciferase as a reporter of environmental regulation of gene expression in mycobacteria.
Google Scholar Crossref. Search ADS. Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein.
Activity of mycobacterial promoters during intracellular and extracellular growth. Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria.
Escherichia coli -mycobacteria shuttle vectors for operon and gene fusions to lac Z: the pJEM series. Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses.
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