A high-throughput approach to promoter study using green fluorescent protein.
Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic. By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP. In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates. Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis. In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats. Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose. Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates. They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time. These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format.
Lu
Canghai
C
Center for Advanced Sensor Technology, Department of Chemical and Biochemical Engineering, University of Maryland-Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Bentley
William E
WE
Rao
Govind
G
D000154:Aconitate Hydratase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005240:Feasibility Studies; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C095500:SodA protein, Bacteria; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase