生物超微弱发光的光子计数成象方法

介绍了我们研制和设计的用于测量生物超微弱发光的光子计数成象系统，包括光子成象头，高帧频ＣＣＤ摄象机，图象采集系统和计算机，可以定量地探测出生物的发光强度分布 ，还能够显示出其发光的图象，确定发光的形状和位置，本系统的信噪比为１０：１，最小探测功率达１０＾－１６Ｗ，灵敏度高，动态范围大。     

Assessing wastewater metal toxicity with bacterial bioluminescence in a bench-scale wastewater treatment system

The effectiveness of a previously developed toxicity monitoring method for activated sludge wastewater treatment employing a bioluminescent bacterium (Shk1) was evaluated in batch experiments and a bench-scale activated sludge system exposed to heavy metals (Cu, Zn, Ni, and Cd). Influent wastewater (primary clarifier supernatant) and activated sludge from a municipal wastewater treatment plant were used in both batch experiments and in the bench-scale wastewater treatment system. Shk1 bioluminescence was most sensitive to Cd and Zn, followed by Cu, and then Ni in order of decreasing sensitivity. In contrast, activated sludge specific oxygen uptake rate was most sensitive to Cu, followed by Cd and Zn, and finally Ni. The same pattern of sensitivity was observed in batch and bench-scale evaluations. Batch experiments examining the effect of metal adsorption were performed. The adsorption of metals to activated sludge and reduction in bioavailability due to chelation by soluble organics or by precipitation in wastewater was found to be an important effect in mediating differences in toxicity response between bioluminescence and respirometry. Batch adsorption experiments indicated that the activated sludge adsorption capacity was highest for Cu, followed by Cd, Ni, and then Zn. A simple mathematical model for the soluble metal concentration in the aeration basin and clarifier was developed utilizing metal distribution coefficients determined from the batch adsorption experiments. Model predictions compared well with results from the bench-scale activated sludge experiments.     

Numerical approximation of bioluminescence tomography based on a new formulation

Bioluminescence tomography (BLT) is a promising new method in biomedical imaging. The BLT problem is an ill-posed inverse source problem, usually studied through a regularization technique. A new approach is proposed for solving the BLT problem based on an adjoint equation. Numerical examples show that the new formulation allows us to obtain accurate solutions.     

Development of an RNA detection system using bioluminescence resonance energy transfer

Here we demonstrate a new RNA detection system using bioluminescence resonance energy transfer (BRET). Recombinant proteins were constructed consisting of enhanced yellow fluorescence protein (EYFP) and Renilla luciferase (RLuc) tethered by Arginine Rich Motif (ARM) peptides, which recognize specific RNA motifs and change their conformation upon binding to them. Conformational transitions of ARM peptides upon RNA binding then trigger a change in the BRET signal between RLuc and EYFP. Additionally, in order to detect arbitrarily selected RNAs, split-RNA probes were designed to reconstruct the ARM peptide binding motif by hybridizing with the target RNA. Combination of BRET probes and split-RNA probes enables to choose and detect target RNA sequences.     

Conditions required for the stimulation of bioluminescence activity of the genetically engineered bacteria, P. putida mt-2 KG1206, preserved by deep-freezing

Herein the conditions required for the stimulation of bioluminescence activity in a genetically engineered strain of Pseudomonas putida mt-2 KG1206, containing the intact TOL plasmid and a constructed plasmid with the P_m-lux gene, are reported upon. Both sodium lactate (SL) and potassium nitrate (KNO₃) were able to stimulate the bioluminescence activity, but a greater increase was observed with nitrogen amendment. This selected stimulant was then tested on reconstituted cells that had been preserved by deep-freezing and mixed with pure inducer solution or groundwater samples. The stimulation of bioluminescence activities for deep-frozen strain was in the range of 101-238% of the control. The effect of KNO₃ was found to be dependent on the type of inducers used and the cell conditions. In general, high bioluminescence activity was observed with groundwater samples, contaminated with high inducer compounds. However, no significant correlation was observed between the bioluminescence intensity and the total inducer concentration in the environmental samples contaminated with complex mixtures with inducers.These results should be useful when other recombinant bioluminescence strains are to be used for environmental monitoring. Overall, the results of this study demonstrate the stimulant conditions for the bioluminescence activity of genetically engineered bacteria, and suggest the potential for preliminary application of this deep-frozen engineered strain in a field-ready bioassay to conveniently detect or monitor a specific group of environmental contaminants.     

Identification of β-lactam antibiotics using bioluminescent Escherichia coli and a support vector machine classifier algorithm

We propose the use of bioluminescent whole cell biosensor combined with a pattern classification algorithm to automatically detect and identify β-lactam antibiotic substances. Escherichia coli cells with a plasmid harboring luxCDABE genes under the β-lactam sensitive promoter element are used as sensors. We present experimental measurements of light production of bioluminescent bacteria subject to 11 antibiotic substances. The patterns of measured light production are classified using a support vector machine classifier. The accuracy and reliability of the classification suggests that this method can be used in the future to probe for new antibiotic substances.     

RAPID METHODS FOR THE DETECTION OF YEAST AND LACTOBACILLUS BY ATP BIOLUMINESCENCE

Present plating methods for the detection of potential spoilage organisms in packaged beers require 2–3 days for yeast, and 5–7 days for lactic acid bacteria. Simple and inexpensive sterility tests based on bioluminescence have been developed for the detection in broth media of small numbers of yeasts in 1 day, and lactic acid bacteria, in both normal and heat-stressed conditions, in 3 days.     

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.     

High hydrostatic pressure effects on bacterial bioluminescence

The mechanism that leads to microbial inactivation by high hydrostatic pressure remains elusive. In this study, a high-pressure system interfaced with a photomultiplier tube (PMT) was developed to monitor cellular metabolism in situ using bioluminescent bacterial strains. Preliminary characterization of the system was performed using Pseudomonas fluorescens 5RL expressing lux proteins from Vibrio fischeri. Stepwise increases in pressure at 34 MPa and above resulted in decreased bioluminescence. Square wave exposure to pressures of 69, 103 and 138 MPa showed bioluminescence reductions greater than 95%, but when cells were returned to ambient pressure bioluminescence returned to 51, 38, and 4% of initial bioluminescence values, respectively. An Escherichia coli strain expressing lux proteins from V. fischeri was constructed to determine whether this reversible effect could be observed in another bacterial genus. Square wave perturbations of 69, 103 and 138 MPa resulted in bioluminescence reductions of about 94% at the highest pressure treatment. Upon decompression, bioluminescence returned to 74, 58 and 30% of the initial bioluminescence values for cells treated at 69, 103 and 138 MPa, respectively. These results suggest that square wave exposure to pressure up to 138 MPa induces reversible cell damage in P. fluorescens 5RL and E. coli VF lux.     

Detection of furfural and 5-hydroxymethylfurfural with a yhcN::luxCDABE bioreporter strain

RT-qPCR data demonstrated that the yhcN gene was highly expressed when Escherichia coli was exposed to 500 ppm of ferulic acid (9.8-fold), vanillin (7.3-fold) or furfural (2.2-fold). Consequently, an E. coli yhcN::lux bioreporter strain (DMY1) was constructed. This strain is very responsive to furfural and 5-hydroxymethylfurfural, showing a maximum induced bioluminescence of 29.4- and 17.8-fold, respectively. The responses to different phenolics were also significant, with relative responses of between 4- and 16-fold. In tests with rice straw hydrolysate samples, a dual induction of DMY1 was observed and it was demonstrated that this corresponded to the activities of the phenolics and HMF within the sample. The maximum response seen with the hydrolysate sample was 3.5 and the minimum phenolic concentration detected was 39 mg/L. These results illustrate that this strain can be used to monitor for the presence of furans and phenolics commonly found within plant hydrolysate samples.