Broad-spectrum protein biosensors for class-specific detection of antibiotics

Abstract

The dramatically increasing prevalence of multi-drug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming. Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics. We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values. This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format. At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics. Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

“…3A), established using the same parameters, and defined phloretin concentrations. Similarly, the half-life of phloretin was estimated based on the degradation dynamics observed in cell culture (61,62).…”

A synthetic mammalian gene circuit reveals antituberculosis compounds

“…pWW491 (O ethR-P hsp70min-SEAP-pA) was obtained by direct cloning of a synthetic OethR sequence (5Ј-gacgtcgatccacgctatcaacgtaatgtcgaggccgtcaacgagatgtcgacactatcgacacgtagcctgcagg-3Ј) (AatII/SbfI) into pMF172 (27). pWW488 (P T7-ethR-vp16-his 6) was constructed by PCR-mediated amplification of ethR-vp16 from pWW489 by using oligonucleotides OWW400 and OWW60 (5Ј-gctctagagcaagcttttaatggtgatggtgatgatgcccaccgtactgtcaattccaag-3Ј) followed by cloning (NdeI/HindIII) into pRSETmod (28). pWW856 (P ethR-gfp-pA) was constructed in three steps: (i) gfp was PCR-amplified from pLEGFP-N1 (Clontech) by using oligonucleotides OWW848 (5Ј-ggcttgaattcaaaggagatataccatggtgagcaagggcgag-3Ј) and OWW849 (5Ј-ggctttctagacaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttacttgtacagctcgtccatgccg-3Ј) and cloned (EcoRI/XbaI) into pWW56 (27) (pWW854).…”

“…pWW871 (5ЈLTR-⌿ ϩ -ethRvp16-P PGK-neo R -3ЈLTR) was designed by cloning ethR-vp16 of pWW489 (EcoRI/ BamHI) into pMSCVneo (Clontech). pWW35 (P SV40-E-vp16-pA), pWW37 (ETR-P hCMVmin-seap-pA) and pWW313 (PT7-E-his6-pA) have been described (28).…”

Pharmacologic transgene control systems for gene therapy

“…A panoply of different inducible mammalian cellcompatible gene regulation systems was developed in the past decade [1,2], which have been used successfully in almost all areas of basic and applied life science research, such as (i) model in vitro and in vivo gene therapy studies (see below; [2,21,25]); (ii) prototype biopharmaceutical manufacturing [11,[26][27][28][29]; (iii) tissue engineering [2,21,30]; (iv) drug discovery [31,32]; (v) functional genomic research [33,34]; and (vi) manifold basic research applications [35][36][37]. Despite their excellent regulation performance, most available transgene control modalities are incompatible with human therapeutic use since the inducer is not clinically licensed or shows unsuited pharmacokinetics [38][39][40][41].…”