A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter.
A cell-free biosensor for the detection of transcription induction by specific small-molecule ligands is presented. As model systems, tetracycline and mercury-inducible promoters were used containing firefly luciferase as reporter gene. Escherichia coli S30 extract was prepared and used for coupled transcription-translation reactions. By using purified Tet repressor and MerR regulatory proteins, we could study repressor-operator interactions for optimizing the relative concentrations of each component. Previously, detection of tetracycline and mercury using similar transcriptional regulation in whole living cells has been carried out. As compared to whole-cell biosensors, our results showed better sensitivity for the detection of tetracycline and the toxic effect of mercury was avoided in the cell-free system. Also, as the system omits cell cultivation and bacterial membranes as molecule passage inhibitors, it is possible to carry out assays in much shorter times and without the use of genetically modified organisms.
Pellinen
Teijo
T
Department of Biochemistry and Food chemistry, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland.
Huovinen
Tuomas
T
Karp
Matti
M
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques; D002474:Cell-Free System / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D008628:Mercury / Q000032:analysis*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry*; D013754:Tetracyclines / Q000032:analysis*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; D049409:Luciferases, Firefly; D008628:Mercury