Differential rates of gene expression monitored by green fluorescent protein.
The use of green fluorescent protein (GFP) as a reporter gene has made a broad impact in several areas, especially in studies of protein trafficking, localization, and expression analysis. GFP's many advantages are that it is small, autocatalytic, and does not require fixation, cell disruption, or the addition of cofactors or substrates. Two characteristics of GFP, extreme stability and chromophore cyclization lag time, pose a hindrance to the application of GFP as a real-time gene expression reporter in bioprocess applications. In this report, we present analytical methods that overcome these problems and enable the temporal visualization of discrete gene regulatory events. The approach we present measures the rate of change in GFP fluorescence, which in turn reflects the rate of gene expression. We conducted fermentation and microplate experiments using a protein synthesis inhibitor to illustrate the feasibility of this system. Additional experiments using the classic gene regulation of the araBAD operon show the utility of GFP as a near real-time indicator of gene regulation. With repetitive induction and repression of the arabinose promoter, the differential rate of GFP fluorescence emission shows corresponding cyclical changes during the culture.
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, 21250, USA.
Canghai
C
Lu
C Renee
CR
Albano
William E
WE
Bentley
Govind
G
Rao
D001089:Arabinose / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002701:Chloramphenicol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005240:Feasibility Studies; D005285:Fermentation; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005819:Genetic Markers; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics*; D009876:Operon; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D001426:Bacterial Proteins; D005819:Genetic Markers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D002701:Chloramphenicol; D001089:Arabinose; D005947:Glucose