Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.     

Symbiosis-stage associated alterations in quorum sensing autoinducer molecules biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti strains use several N-acylhomoserine lactone (AHL) autoinducers to sense the population density during saprophytic growth. Using a sensitive gfp-based AHL biosensor system, we show that synthesis of short-chain AHL molecules is inhibited (or significantly diminished) during the symbiotic phase of growth and is undetectable in the nitrogen fixing bacteroids. In the saprophytic zone V of nodules occupied by intensively proliferating vegetative forms of rhizobia, AHL production recommenced and bright-green fluorescence was observed concomitantly with increasing population density.     

Isolation of the crude oil degrading marine Acinetobacter sp. E11

The Acinetobacter sp. E11, isolated from Port Dickson Beach, Malaysia, was able to grow in media containing crude oil as the sole carbon and energy source. Substrate specificity studies showed that the bacterium exhibited substrate preference as growth was observed only in media containing aliphatic hydrocarbons, while aromatic and cyclic hydrocarbons inhibited growth. With the aliphatic hydrocarbons, growth was seen only in the long-chain alkanes tested (pentadecane, dodecane and hexadecane). No growth was recorded in the short-chain alkanes (pentane, hexane and heptane) tested. With complex hydrocarbons, only crude oil and 4T SHELL engine oil supported growth. No growth was observed in kerosene and PETRONAS gasoline. The isolate could grow in up to 10% and 20% [v/v] of the crude oil and alkanes tested, respectively. Among the long-chain alkanes tested, hexadecane was the most preferred, followed by pentadecane and dodecane. Nitrogen and phosphorous supplements were essential for growth and the best growth was achieved with 3% nitrogen/phosphorous additions. Microscopic observation revealed that the bacterium adhered to the hexadecane and crude oil droplets. GC analysis showed that the bacterium was able to degrade more than 60% of the hydrocarbons in the crude oil in 15 days at 37°C compared to the uninoculated media.     

Application of Transcription Factor-based 3-Hydroxypropionic Acid Biosensor

Application of the recently developed wholecell 3-HP biosensor, which detects 3-hydroxypropionic acid (3-HP) and expresses fluorescence as an output signal in response to the 3-HP concentration, was studied in three areas of enzyme and metabolic engineering. First, a sensor was applied to identify active 3-hydroxyaldehyde dehydrogenase (ALDH), a key enzyme in the 3-HP production pathway. Second, with the aid of helper enzymes that catalyze the conversion of 1,3-propanediol (1,3-PDO) to 3-HP, a 3-HP biosensor was converted into a 1,3-PDO biosensor. Third, a 3-HP biosensor, with proper modifications in expression module of the output signal and the use of pH-tolerant red fluorescent protein (RFP), was shown to monitor the rate of 3-HP production under process conditions in which one or more interfering compounds are present in the culture medium and/or the medium pH decreases. This study demonstrates that 3-HP biosensors can be widely used in enzyme and metabolic engineering applications for 3-HP production.     

A facile fluorescence turn-on biosensor customized for monitoring of protein kinase activity based on carboxylic carbon nanoparticle–peptide complexes

In this study, we present a facile and low-cost approach for detecting protein kinase A (PKA) by assembling a purpose-designed carboxyfluorescein (FAM)-labelled peptide with carboxylic carbon nanoparticles (CNPs). Fluorescence of the FAM-labelled peptide gradually decreases to a low background signal as a result of the electron transfer from CNPs to FAM-labelled peptide via the peptide, which acts as a bridge. The reaction in the sensor in the presence of adenosine 5′-triphosphate and PKA phosphorylates the substrate peptide and disrupts the electrostatic repulsive force between the CNPs and the peptide, therefore altering the spectroscopic signal of the system. The change in fluorescence signal was directly proportional to the PKA concentration in the range 0–1.8 U/ml with a detection limit of 0.04 U/ml. These results suggest that PKA activity can be effectively measured using the developed PKA biosensor. Moreover, the fluorescence biosensor was successfully used in the investigation of PKA in spiked human embryonic kidney (HEK) 293 cells lysates, indicating its potential applications in protein kinase-related biochemical fundamental research.     

Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL

Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.     

Modular and selective biosynthesis of gasoline-range alkanes

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement.     

Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

Cell signaling systems transmit information by post-translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-protein coupled receptor (GPCR). We used published mass spectrometry-based proteomics data to identify putative sites of phosphorylation on pheromone pathway components, and we used evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of putative phosphorylation events that contribute to adjust the input-output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results suggest that relatively small quantitative influences from individual phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.     

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.     

Cholinesterase biosensor construction - a review

Biosensors using cholinesterases as the biorecognition component have been used to assay organophosphates and carbamates for a long time. In this review, some strategies convenient for biosensor construction are presented. Solutions for cholinesterase immobilization and output signal monitoring are presented as the basic presumptions for successful biosensor construction.     

Optimizing the cationic conjugated polymer-sensitized fluorescent signal of dye labeled oligonucleotide for biosensor applications

Methods for real time, highly selective and sensitive polynucleotide detection are of vast scientific and economic importance. Fluorescence resonance energy transfer (FRET)-based assays which take advantage of the collective response of water-soluble conjugated polymers (CPs) and the self-assembly characteristic of aqueous polyelectrolytes have been widely used for the detection of DNA, RNA, protein and small molecules. The detection sensitivity of CP-based biosensor is dependent on the signal amplification of dye emission upon excitation of CP relative to that upon direct excitation of the dye. Using cationic polyfluorene derivatives and chromophore (fluorescein or Texas Red) labeled single-stranded DNA molecules (ssDNA-C*) as donor/acceptor pairs, we show that in addition to the spectral overlap, orientation and distance between the donor and the acceptor, the energy levels and fluorescence quenching of the donor/acceptor within the polymer/DNA-C* complexes are also important factors that affect the signal output of dye emission.     

A fiber optic biosensor for fluorimetric detection of triple-helical DNA.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.     

Incorporation and localisation of alkanes in a protomembrane model by neutron diffraction

Protomembranes at the origin of life were likely composed of short-chain lipids, readily available on the early Earth. Membranes formed by such lipids are less stable and more permeable under extreme conditions, so a novel membrane architecture was suggested to validate the accuracy of this assumption. The model membrane includes the presence of a layer of alkanes in the mid-plane of the protomembrane in between the two monolayer leaflets and lying perpendicular to the lipid acyl chains. Here, we investigated such a possibility experimentally for membranes formed by the short-chain phospholipid 1,2-didecanoyl-sn-glycero-3-phophocholine, including or not the alkanes eicosane, squalane or triacontane by means of neutron membrane diffraction and contrast variation. We found strong indications for incorporation of two of the three alkanes in the membrane mid-plane through the determination of neutron scattering length density profiles with hydrogenated vs deuterated alkanes and membrane swelling at various relative humidities indicating a slightly increased bilayer thickness when the alkanes are incorporated into the bilayers. The selectivity of the incorporation points out the role of the length of the n-alkanes with respect to the capacity of the membrane to incorporate them.     

Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

Brain cell metabolism is intimately associated with intracellular oxidation–reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca²⁺ abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.     

A novel approach to improve specificity of algal biosensors using wild-type and resistant mutants: an application to detect TNT

A new genetic approach was developed for increasing specificity of microalgal biosensors. This method is based on the use of two different genotypes jointly to detect a given pollutant: (i) a sensitive genotype to obtain sensitivity; and (ii) a resistant mutant to obtain specificity. The method was tested by the development of a microalgal biosensor for the detection of the explosive 2,4,6-trinitrotoluene (TNT) using a wild-type strain (DcG1wt) of Dictyosphaerium chlorelloides (Chlorophyceae) as the sensitive organism, and a TNT-resistant mutant, obtained from DcG1wt strain by a modified Luria-Delbrück fluctuation analysis. The inhibition of chlorophyll a fluorescence of PSII by TNT was used as the biological signal. Significant differences in maximal fluorescence of light-adapted algae (F'(m)) between wild-type DcG1wt cells and TNT-resistant mutants, were observed in all the TNT concentrations tested (from 0.5 to 31.3 mg l(-1)) after only 3 min of exposure. Resistant mutants always exhibited significant higher F'(m) values in the presence of TNT than wild-type cells. These results suggest that the use of two different genotypes (sensitive and resistant to a given pollutant) jointly is a useful method to improve microalgal biosensors specificity.     

Ratiometric fluorescence imaging of dual bio-molecular events in single living cells using a new FRET pair mVenus/mKOκ-based biosensor and a single fluorescent protein biosensor

Genetically coded fluorescent protein (FP)-based biosensors are powerful tools for the non-invasive tracking of molecular events in living cells. Although a variety of FP biosensors are available, the simultaneous imaging of multiple biosensors (multi-parameter imaging) in single living cells remains a challenge and is far from routinely used to elucidate the intricate networks of molecular events. In this study, we established a novel combination of FP biosensors for dual-parameter ratiometric imaging, consisting of a new fluorescence resonance energy transfer (FRET) pair mVenus (yellow FP)/mKOκ (orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor Grx1-roGFP2. Under our imaging condition, 1.4±0.05% of Grx1-roGFP2 signal contributes to the mVenus channel and 5.2±0.12% of the mVenus signal contributes to the Grx1-roGFP2 channel. We demonstrate that such low degree of cross-talk causes negligible distortion of the ratiometric signal of the YO-based FRET biosensor and Grx1-roGFP2. By using this dual-parameter ratiometric imaging approach, we achieved simultaneous imaging of Src/Ca(2+) signaling and glutathione (GSH) redox potential in a single cell, which was previously unattainable. Furthermore, we provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H(2)O(2) via its effect on GSH-based redox system, demonstrating the power of this dual-parameter imaging approach for elucidating new connections between different molecular events that occur in a single cell. More importantly, the dual-parameter imaging approach described in this study is highly extendable.