Integrating porphyrin-based nanoporous organic polymers with electrochemical aptasensors for ultratrace detection of kanamycin.

The synthesis and characterization of two new porphyrin-based porous organic polymers (POPs) via Sonogashira cross-coupling reaction and leverage the two obtained POPs is reported for the fabrication of electrochemical aptasensors to detect kanamycin at an ultratrace level. The resultant electrochemical aptasensor demonstrates a high linear relationship with the logarithmic value of kanamycin concentration in the range 5 × 10-5-5 µg/L with the limit of detection of 17.6 pg/L or 36.3 fM. During the analysis of real samples from milk and river, a relative standard deviation of less than 4.39%, and good recovery values in the range 97.0-105% were obtained.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli.

BACKGROUND: The aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq). RESULTS: Over two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing. CONCLUSIONS: The results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Diadenosine tetraphosphate modulated quorum sensing in bacteria treated with kanamycin.

BACKGROUND: The dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress. RESULTS: Here, we labeled Ap4A with biotin and incubated the labeled Ap4A with the total proteins extracted from kanamycin-treated Escherichia coli to identify the Ap4A binding protein in bacteria treated with kanamycin. Liquid chromatography‒mass spectrometry (LCMS) and bioinformatics were used to identify novel proteins that Ap4A interacts with that are involved in biofilm formation, quorum sensing, and lipopolysaccharide biosynthesis pathways. Then, we used the apaH knockout strain of E. coli K12-MG1655, which had increased intracellular Ap4A, to demonstrate that Ap4A affected the expression of genes in these three pathways. We also found that the swarming motility of the apaH mutant strain was reduced compared with that of the wild-type strain, and under kanamycin treatment, the biofilm formation of the mutant strain decreased. CONCLUSIONS: These results showed that Ap4A can reduce the survival rate of bacteria treated with kanamycin by regulating quorum sensing (QS). These effects can expand the application of kanamycin combinations in the treatment of multidrug-resistant bacteria.

High efficient electrochemical biosensor based on exonuclease-â ¢-assisted dual-recycling amplification for ultrasensitive detection of kanamycin.

A target-triggered and exonuclease-Ⅲ-assisted strand displacement, dual-recycling amplification reaction-based biosensor was developed for the rapid, ultrasensitive and accurate detection of kanamycin. The robust profiling platform was constructed using high conductive MXene/VS2 for the electrode surface modification and high active CeCu2O4 bimetallic nanoparticles as nanozyme to improve the sensitivity as well as the catalytic signal amplification of the biosensor. Using the dual supplementary recycling of primer DNA and hairpin DNA, the electrochemical platform could accurately detect kanamycin to as low as 0.6 pM from the range of 5 pM to 5 µM. By profiling five other antibiotics, this platform exhibited high specificity, enhanced repeatability and reproducibility. Based on these intrinsic characteristics and by utilizing milk and water samples, the as-designed biosensor offers a remarkable strategy for antibiotic detection due to its favorable analytical accuracy and reliability, thereby demonstrating potential application prospect for various antibiotic biosensing in food quality control, water contamination detection and biological safety analysis.

Deglycosylation Inactivation Initiated by a Novel Periplasmic Dehydrogenase Complex Provides a Novel Strategy for Eliminating the Recalcitrant Antibiotic Kanamycin.

Biodegradation using enzyme-based systems is a promising approach to minimize antibiotic loads in the environment. Aminoglycosides are refractory antibiotics that are generally considered non-biodegradable. Here, we provide evidence that kanamycin, a common aminoglycoside antibiotic, can be degraded by an environmental bacterium through deglycosylation of its 4'-amino sugar. The unprecedented deglycosylation inactivation of kanamycin is initiated by a novel periplasmic dehydrogenase complex, which we designated AquKGD, composed of a flavin adenine dinucleotide-dependent dehydrogenase (AquKGDα) and a small subunit (AquKGDγ) containing a twin-arginine signal sequence. We demonstrate that the formation of the AquKGDα-AquKGDγ complex is required for both the degradation activity of AquKGD and its translocation into the periplasm. Native AquKGD was successfully expressed in the periplasmic space of Escherichia coli, and physicochemical analysis indicated that AquKGD is a stable enzyme. AquKGD showed excellent degradation performance, and complete elimination of kanamycin from actual kanamycin manufacturing waste was achieved with immobilized AquKGD. Ecotoxicity and cytotoxicity tests suggest that AquKGD-mediated degradation produces less harmful degradation products. Thus, we propose a novel enzymatic antibiotic inactivation strategy for effective and safe treatment of recalcitrant kanamycin residues.

Label-free "signal-off" PEC aptasensor for determination of kanamycin based on 3D nanoflower-like FeIn2S4/CdS Z-scheme heterostructures.

Highly photoactive 3D nanoflower-like FeIn2S4/CdS heterostructures were synthesized by hydrothermal treatment and low-temperature cation exchange. The FeIn2S4/CdS displayed 14.5 times signal amplification in contrast to FeIn2S4 alone. It was applied as a photoactive substrate to construct a label-free photoelectrochemical (PEC) aptasensor for ultrasensitive determination of kanamycin (KAN). Under the optimal conditions, the constructed PEC aptasensor displayed a wide linear range (5.0 × 10-4 ~ 5.0 × 101 ng mL-1) and a low detection limit (S/N = 3) of 40.01 fg mL-1. This study provides some constructive insights for preparation of advanced photoactive materials and exhibits great potential for quantitative determination of antibiotics in foods and environmental samples.

Sub-femtomolar capacitance-based biosensing of kanamycin using screen-printed electrodes coated with redox-active polymeric films.

An ultrasensitive capacitance-based biosensor has been developed capable of detecting the kanamycin (KAN) antibiotic at sub-femtomolar levels. The biosensor was constructed using a potential-pulse-assisted method, allowing for the layer-by-layer deposition of a melanin-like polymeric film (MLPF) on an electrode surface modified with gold nanoparticles (AuNPs). The MLPF was formed through the electrochemical polymerization of dopamine and the specific kanamycin aptamer. By optimizing the operating parameters, we achieved a label-free detection of kanamycin by monitoring the variation of pseudocapacitive properties of the MLPF-modified electrode using electrochemical impedance spectroscopy. The developed biosensor demonstrated a wide linear response ranging from 1 fM to 100 pM, with a remarkable limit of detection of 0.3 fM (S/N = 3) for kanamycin. Furthermore, the biosensor was successfully applied to detect kanamycin in milk samples, exhibiting good recovery. These findings highlight the promising potential of the aptasensor for determination of antibiotic residues and ensuring food safety. In conclusion, our ultrasensitive capacitance-based biosensor provides a reliable and efficient method for detecting trace amounts of kanamycin in dairy products. This technology can contribute to safeguarding consumer health and maintaining high food safety standards.

An unusual complication secondary to kanamycin use in a patient of multidrug-resistant pulmonary tuberculosis.

Gitelman syndrome (GS) is a rare genetic renal disease characterized by hypomagnesemia, hypokalemia, hypocalciuria, and metabolic alkalosis. It usually presents in late childhood or early adulthood. A 30-year-old female diagnosed case of multidrug-resistant (MDR-TB) pulmonary tuberculosis 2 months ago presented to our outpatient department with intermittent painful spasms in all four limb muscles. Her treatment regimen consisted of kanamycin, levofloxacin, cycloserine, and ethionamide. On further evaluation, her investigations revealed hypokalemia, hypocalcemia, hypomagnesemia, metabolic alkalosis with normal serum creatinine level. She was initially treated with intravenous calcium and potassium. However, the electrolyte abnormalities and metabolic alkalosis persisted. All her lab parameters became normal after discontinuing kanamycin and electrolyte replacement for 4 weeks. She was discharged and advised to continue her antituberculosis treatment. There was no recurrence of symptoms on further follow up.

Distribution of Common and Rare Genetic Markers of Second-Line-Injectable-Drug Resistance in Mycobacterium tuberculosis Revealed by a Genome-Wide Association Study.

Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.

Target-mediated competitive hybridization of hairpin probes for kanamycin detection based on exonuclease III cleavage and DNAzyme catalysis.

Based on aptamer recognition and target-mediated competitive hybridization of hairpin probes, we developed a fluorescence sensor for kanamycin (KAN) detection. The aptamer and KAN binding will open hairpin H1 to release the trigger DNA fragment, which can initiate the competitive hybridization between hairpins H2 and H3. Then, exonuclease III (Exo III) can cleave H2 and H3 to produce numerous DNA3 and DNA4. Through the synergetic hybridization among DNA1, DNA2, DNA3, and DNA4, an active Mg2+-DNAzyme can be formed. The cleavage reaction toward FAM-BHQ-modified DNA2 will produce a high fluorescence signal for KAN assay. Through Exo III-guided cleavage and Mg2+-DNAzyme-based catalysis, the sensor exhibits high sensitivity, with a detection limit of 3.1 fM. This method is robust and has been applied to the detection of KAN in milk and water samples with good accuracy and reliability. Our developed fluorescence sensor exhibits the advantages of simple operation, high sensitivity, and good robustness, which are beneficial for KAN detection in food samples.

Development of label-free fluorescent biosensor for the detection of kanamycin based on aptamer capped metal-organic framework.

The abuse of antibiotics has caused serious threat to human health, so it is of great significance to develop a simple and sensitive method for the detection of trace residues of antibiotics in the environment and food. Herein, a novel label-free fluorescent biosensing platform based on the fluorescence change of aptamers-capped zeolitic imidazolate framework-8 (ZIF-8) @ 2,2',2″,2‴-((ethene-1,1,2,2-tetrayltetrakis (benzene-4,1-diyl)) tetrakis (oxy)) tetraacetic acid (TPE) through ATP-assisted competitive coordination reaction was designed for such an end. ZIF-8@TPE/Aptamer (Apt) emits strong fluorescence at 425 nm in HEPES buffer due to the aggregation induced luminescence properties of TPE molecules in confined state. Once kanamycin was added, the conformation of aptamer capped on the surface of ZIF-8@TPE changes because of the specific recognition of kanamycin with aptamer, leading to the collapse of ZIF-8 and release of TPE, accompanied with a dramatic decrease of fluorescence intensity. Under the optimal conditions, a good correlation was obtained between the fluorescence intensity of ZIF-8@TPE/Apt and the concentration of kanamycin ranging from 10 to 103 ng/mL with a detection limit of 7.3 ng/mL. The satisfactory analytical performance of the assay for kanamycin detection suggests good prospect for its application in food safety analysis.

Aminoglycoside treatment alters hearing-related genes and depicts behavioral defects in Drosophila.

The hearing organ of Drosophila is present within the second segment of antennae. The hearing organ of Drosophila (Johnston's organ [JO]) shares much structural, developmental, and functional similarity with the vertebrate hearing organ (Organ of Corti). JO is evolving as a potential model system to examine the hearing-associated defects in vertebrates. In the vertebrates, aminoglycosides like gentamicin, kanamycin, and neomycin have been known to cause defects in the hearing organ. However, a complete mechanism of toxicity is not known. Taking the evolutionary conservation into account the current study aims to test various concentrations of aminoglycoside on the model organism, Drosophila melanogaster. The current study uses the oral route to check the toxicity of various aminoglycosides at different concentrations (50, 100, 150, 200, and 250 µg ml- 1 ). In Drosophila, many foreign particles enter the body through the gut via food. The aminoglycoside treated third instar larvae show defective crawling and sound avoidance behavior. The adult flies release lower amounts of acetylcholine esterase and higher amounts of reactive oxygen species than control untreated animals, accompanied by defective climbing and aggressive behavior. All these behavioral defects are further confirmed by the altered expression level of hearing genes such as nompC, inactive, nanchung, pyrexia. All the behavioral and genetic defects are reported as a readout of aminoglycoside toxicity.

Single-step antibiotic-mediated synthesis of kanamycin-conjugated gold nanoparticles for broad-spectrum antibacterial applications.

Widespread and irrational use of antibiotics results in the development of antibiotic-resistant bacteria. Thus, there is a need to develop novel antibacterial agents in order to replace conventional antibiotics and to increase the efficacy of already existing antibiotics by combining them with other materials. Herein, a single-step antibiotic-mediated synthesis of antibiotic-conjugated gold nanoparticles is reported. In this single-step method antibiotic Kanamycin, an aminoglycoside itself plays the role of reducing as well as capping agent by reducing gold salt into gold nanoparticles. The kanamycin-conjugated gold nanoparticles (Kan-AuNPs) were confirmed by UV-Visible spectroscopy and further physico-chemically characterized by various instrumental techniques. Synthesized Kan-AuNPs showed broad-spectrum antibacterial activity against Gram-positive Staphylococcus aureus as well as Gram-negative Escherichia coli bacterial strains. They are also found to be effective against Pseudomonas aeruginosa and pathogenic E. coli isolated from urinary tract infections (UTIs) patients, which are responsible to cause hospital-acquired infections like nosocomial, burn wound and UTIs. The minimum inhibitory concentration (MIC) of Kan-AuNPs is 50 µg ml-1 for S. aureus and E. coli, 125 µg ml-1 for P. aeruginosa and 100 µg ml-1 for E. coli isolated from UTIs patients. It is also evident that the MIC of Kan-AuNPs for antibacterial activity is lower as compared to antibiotic kanamycin alone for all bacterial strains. Hence, the one-step strategy of synthesis for Kan-AuNPs is a suitable strategy for fighting infectious bacterial strains in hospitals, healthcare and the pharmaceutical industry.

Alarmone Ap4A is elevated by aminoglycoside antibiotics and enhances their bactericidal activity.

Second messenger molecules play important roles in the responses to various stimuli that can determine a cell's fate under stress conditions. Here, we report that lethal concentrations of aminoglycoside antibiotics result in the production of a dinucleotide alarmone metabolite-diadenosine tetraphosphate (Ap4A), which promotes bacterial cell killing by this class of antibiotics. We show that the treatment of Escherichia coli with lethal concentrations of kanamycin (Kan) dramatically increases the production of Ap4A. This elevation of Ap4A is dependent on the production of a hydroxyl radical and involves the induction of the Ap4A synthetase lysyl-tRNA synthetase (LysU). Ectopic alteration of intracellular Ap4A concentration via the elimination of the Ap4A phosphatase diadenosine tetraphosphatase (ApaH) and the overexpression of LysU causes over a 5,000-fold increase in bacterial killing by aminoglycosides. This increased susceptibility to aminoglycosides correlates with bacterial membrane disruption. Our findings provide a role for the alarmone Ap4A and suggest that blocking Ap4A degradation or increasing its synthesis might constitute an approach to enhance aminoglycoside killing potency by broadening their therapeutic index and thereby allowing lower nontoxic dosages of these antibiotics to be used in the treatment of multidrug-resistant infections.

Non-thiolated nucleic acid functionalized gold nanoparticle-based aptamer lateral flow assay for rapid detection of kanamycin.

A novel Apt-LFA has been established for kanamycin based on non-thiolated nucleic acid-modified colloidal gold nanoprobe (AuNPs@polyA-DNA). The improvement in nucleic acid hybridization speed and efficiency was verified by modifying AuNPs with polyA-DNA strands instead of thiolated oligonucleotides (SH-DNA) strands. Moreover, the AuNPs@polyA-DNA was explored to apply in an Apt-LFA. The experimental factors including the concentration of the aptamer, the concentration of SA-DNAT conjugate, the incubation time, and temperature were carefully investigated. In addition, the kanamycin aptamer was modified by extending several bases at its end to modulate the hybridization complementary strand, which was found to significantly improve the performance of Apt-LFA. Under optimal experimental conditions, the Apt-LFA can detect kanamycin in honey with a LOD of 250 ng mL-1 by the naked eyes. A linear range of 50-1250 ng mL-1 was obtained with a LOD of 15 ng mL-1 in honey by a portable reader. The Apt-LFA was successfully applied to the detection of kanamycin in honey with recoveries of 95.1-105.2%.

Dual DNAzyme-catalytic assembly of G-quadruplexes for inducing the aggregation of gold nanoparticles and developing a novel antibiotic assay method.

By utilizing a target biorecognition reaction to induce the self-assembly of G-quadruplexes and the aggregation of gold nanoparticles (Au NPs), this work develops a novel colorimetric biosensing method for kanamycin (Kana) antibiotic detection. The compact G-quadruplex structure was assembled from its two half-split sequences which were designed in two hairpin substrates of the Mg2+-dependent DNAzyme (MNAzyme). Besides hybridizing with the aptamer strand, the MNAzyme sequence was also split into two half fragments to be designed in the two substrates. Upon the aptamer-recognition reaction toward Kana, the MNAzyme strand could be quantitatively released to cause the exposure of the split G-quadruplex-sequences on two hairpin substrate-modified Au NPs and simultaneous release of two half fragments of the MNAzyme-sequence. Thus, the K+-assisted self-folding of G-quadruplexes causes the cross-linking of the two Au NPs to realize the Au NP aggregation-based colorimetric signal output (measured at the largest absorption peak near 520 nm). Meanwhile, the self-assembled formation of the second MNAzyme drastically amplified the signal response. Under the optimal conditions, a wide linear range from 0.1 pg mL-1 to 10 ng mL-1 and an ultrahigh sensitivity with the detection limit of 76 fg mL-1 were obtained. The dose-recovery experiments in real samples showed satisfactory results with recoveries from 98.4 to 105.4% and relative errors compared with the ELISA method less than 4.1%. Due to the high selectivity, excellent repeatability and stability, and simple manipulation, this method indicates a promising potential for practical applications. A novel homogeneous biosensing method was developed for the convenient detection of the kanamycin antibiotic. The target biorecognition-induced and dual DNAzyme-catalytic assembly of G-quadruplexes enabled the amplified aggregation of gold nanoparticles for the simple, cheap, stable, and ultrasensitive colorimetric signal transduction of the method.

Reusable molecularly imprinted electrochemiluminescence assay for kanamycin based on ordered mesoporous carbon loaded with indium oxide nanoparticles and carbon quantum dots.

A highly sensitive kanamycin electrochemiluminescence (ECL) switch sensor was constructed. A signal element consisting of ordered mesoporous carbon loaded with indium oxide nanoparticles/carbon quantum dots (OMC/In2O3/C-dots) was assembled on the surface of a gold electrode. Then, a molecularly imprinted polymer (MIP) was prepared on the modified electrode surface using kanamycin as the template molecule and o-aminophenol as the functional monomer. After kanamycin elution, the prepared sensor retained specific kanamycin recognition sites. OMC/In2O3 effectively amplified the ECL signal of the C-dots, thereby enhancing the detection sensitivity, whereas kanamycin quenched the signal. Therefore, the imprinted sites acted as a switch, providing a new method for detecting kanamycin. Under the optimal experimental conditions, the concentration of kanamycin was proportional to the degree of ECL quenching within a linear range of 5-4500 × 10-12 mol L-1 at 0.8 V (vs. Ag/AgCl electrode electrode), and the detection limit was 5.8 × 10-13 mol L-1. When applied to the detection of kanamycin in actual samples, such as chicken, duck, pork, and milk, the recovery for spiked samples was in the range 92.7-110%.

Dual-mode photoelectrochemical/electrochemical sensor based on Z-scheme AgBr/AgI-Ag-CNTs and aptamer structure switch for the determination of kanamycin.

A "signal-on" dual-mode aptasensor based on photoelectrochemical (PEC) and electrochemical (EC) signals was established for kanamycin (Kana) assay by using a novel Z-scheme AgBr/AgI-Ag-CNTs composite as sensing platform, an aptamer structure switch, and K3[Fe(CN)6] as photoelectron acceptor and electrochemical signal indicator. The aptamer structure switch was designed to obtain a "signal-off" state, which included an extended Kana aptamer (APT), one immobilized probe (P1), and one blocking probe (P2) covalently linked with graphdiyne oxide (GDYO) nanosheets. P1, P2, and aptamer formed the double helix structure, which resulted in the inhibited photocurrent intensity because of the weak conductivity of double helix layer and serious electrostatic repulsion of GDYO towards K3[Fe(CN)6]. In the presence of Kana, APT specifically bound to the target and dissociated from P1 and P2, and thus, a "signal-on" state was initiated by releasing P2-GDYO from the platform. Based on the sensing platform and the aptamer structure switch, the dual-mode aptasensor realized the linear determination ranges of 1.0 pM-2.0 µM with a detection limit (LOD) of 0.4 pM (for PEC method) and 10 pM-5.0 µM with a LOD of 5 pM (for EC method). The aptasensor displayed good application potential for Kana test in real samples.

Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression.

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5'-untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High-level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.