Establishment Of Tobramycin Whole-cell Microbial Sensor Based On Artificial Ribozyme Switch

Ribozyme,a class of small molecular RNA with catalytic function,integrate with the aptamer screened in vitro to form ribozyme-based switch.The combine of the ligand molecules and aptamers enable the switches catalytic activity through conformation change of ribosyme,allowing the powerful control of the gene expression at the level of transcription or translation.The fact that aminoglycoside antibiotics(AAs)residues are widely found in food and environment,thus result in the high risk for human health for its cycle accumulation in ecology system.The aptasensors,as a new type of aptamer-based biosensors,using aptamer as a biometric element for AAs detection,owing to its satisfactory selectivity,specificity,and sensitivity,have been extensively used in biology or medical industries.In this study,a tobramycin concentration depended whole cell microbial sensor(tob-HHAz)was constructed.As a artificial ribozyme-based switch,it was composed of the tobramycin aptamer and a type of hammerhead ribozyme(HHR)from Schistosoma Mansoni.This tob-HHAz cleavage core would be activate through the special interaction of the tobramycin ligand and aptamer,which allowing anti-RBS sequence release from blocked site and "turn on" expression of the reporter gene e GFP downstream.This aptamer biosensor has been obtained through integrating all the modules into one complete RNA sequence and easily been introduced into Escherichia coli without suffering by harsh external environments,thus realize a non-label,rapid recognition signal detection method in living cells by converting tobramycin concentration into visual fluorescence expression.Here we first constructed an artificial RNA sequence library based on tob-RNA scaffold by randomly designing a key communication module sequence between aptamer and ribozyme,three independent tob-sensitive RNA structures were obtained with high throughput screening in Escherichia coli.Further verification were adapted with vitro shear validation as well as RNA secondary structure bioinformatics analysis,C4 and H6 bacterial sensors were verified.Qualitative and rapid detection of tobramycin in milk samples were performed for verifying the practicability of the assay,both of them exhibited high sensor performance for analysis of sample,with the detection linear range from 0 to 1000 n M,involving the 40 n M minimum detection limit(LODs)(lower than the maximum residue level(MRLs)in milk stipulated by the European Union).In the following qualitative detection of human urine,C4 structure provides a linear range of 30-650 n M with a LODs of 30 n M.Although carried with different aptamers individually,C4 retained high specificity for tobramycin detection as well as H6 in all samples.This whole-cell microbial sensor,regulated protein levels via ribozyme switch,making it feasible for mass-produced by engineered microbial reproduction easily and cheaply.As an living sensor with capacity of stable,sensitive and rapid evaluation of antibiotic,the proprieties of easy application and convenient preservation make it possible to extensively utilize in field detection and real-time diagnosis involving antibiotic analysis.We created a stable and reliable method for building the whole-cell microbial ribozyme-based sensors.For features of variable aptamer region,the sensors provides a general applicable method for further detection of substances ranging from small antibiotic molecules to heavy metal ions,amino acids,proteins etc in living cells,thus enable a great significance to apply these biosensors of the assay in food safety control,environmental monitoring and medical detection.