Development of a histamine aptasensor for food safety monitoring

Histamine produced by bacteria through decarboxylation of histidine in spoiled foods such as fish is known to cause food poisoning. Therefore, accurate and facile measurement of histamine is of practical importance. Using the recently discovered RNA aptamer that specifically recognizes histamine (A1-949 aptamer), we developed an aptasensor based on the structure-switching mechanism. Specifically, the aptamer A1-949 was fluorescently labeled at the 5′ end and hybridized with a short quencher DNA strand that is partially complementary to the aptamer. The quencher strand was modified with a fluorescence quencher at its 3′ terminus. Displacement of the quencher strand upon histamine binding results in an increased fluorescence. After optimizing the assay condition, the enantiomeric version of the aptasensor (L-RNA and L-DNA) was synthesized which could detect the achiral analyte with identical sensitivity and improved biochemical stability. The aptasensor performance was validated by measuring fish samples spiked with known concentrations of histamine. Finally, histamine content in spoiled fish samples was measured, and the results were compared with the measurements using a commercial enzymatic assay kit

Controlling Bdellovibrio bacteriovorus Gene Expression and Predation Using Synthetic Riboswitches

Bdellovibrio bacteriovorus is a predatory bacterium that feeds on Gram-negative bacteria including a wide range of pathogens and thus has potential applications as a biocontrol agent. Owing to its unique life cycle, however, there are limited tools that enable genetic manipulation of B. bacteriovorus. This work describes our first steps toward engineering the predatory bacterium for practical applications by developing basic genetic parts to control gene expression. Specifically, we evaluated four robust promoters that are active during the attack phase of B. bacteriovorus. Subsequently, we tested several synthetic riboswitches that have been reported to function in Escherichia coli, and identified theophylline-activated riboswitches that function in B. bacteriovorus. Finally, we inserted the riboswitch into the bacterial chromosome to regulate expression of the flagellar sigma factor fliA, which was previously predicted to be essential for predation, and observed that the engineered strain shows a faster predation kinetics in the presence of theophylline

Riboswitch Signal Amplification by Controlling Plasmid Copy Number

Riboswitches are cis-acting RNA devices in mRNAs that control gene expression in response to chemical inputs. As RNA aptamers that recognize diverse classes of molecules can be isolated by in vitro selection, synthetic riboswitches hold promise for various applications in synthetic biology. One of the major drawbacks of riboswitches, however, is their limited dynamic range. A high level of gene expression in the OFF state (leakage) is also a common problem. To address these challenges, we designed and constructed a dual-riboswitch plasmid in which two genes are controlled by theophylline-activated riboswitches. One riboswitch controls the gene of interest, and another riboswitch controls RepL, a phage-derived replication protein that regulates the plasmid copy number. This single-plasmid system afforded an ON/OFF ratio as high as 3900. Furthermore, we used the system to control CRISPR interference (CRISPRi) targeting endogenous genes, and successfully observed expected phenotypic changes in Escherichia coli

The Future of Butyric Acid in Industry

In this paper, the different applications of butyric acid and its current and future production status are highlighted, with a particular emphasis on the biofuels industry. As such, this paper discusses different issues regarding butyric acid fermentations and provides suggestions for future improvements and their approaches

HIV-1 CRF 02 AG polymerase genes in Southern Ghana are mosaics of different 02 AG strains and the protease gene cannot infer subtypes

<p>Abstract</p> <p>Background</p> <p>Little is known about the detailed phylogeny relationships of CRF 02_AG HIV-1 polymerase genes in Ghana. The use of the protease gene of HIV-1 for subtyping has shown conflicting results.</p> <p>Methods</p> <p>The partial polymerase gene sequences of 25 HIV-1 strains obtained with Viroseq reagents were aligned with reference subtypes and alignments trimmed to a 300 bp protease, 661 bp and 1005 reverse transcriptase sequence alignments. Phylogenetic relationships of these alignments were determined with the Neighbour-Joining method using 1000 replicates and recombination patterns determined for the sequences with RIP 3.0 in the HIV sequence database.</p> <p>Results</p> <p>Unlike the other alignments, the protease gene had nodes with bootstrap values < 100% for repeat control sequences. Majority of the CRF 02_AG sequences from Ghana were made up of fragments of several strains of CRF 02_AG/AG strains. The protease gene alone is not suitable for phylogenetic analysis.</p> <p>Conclusion</p> <p>The polymerase genes of HIV-1 strains from Ghana are made up of recombinants of several CRF 02_AG strains from Ghana, Senegal and Cameroon, but the clinical implications are unknown. Using the HIV-1 protease gene for subtyping will not infer subtypes correctly.</p

Co-culturing a novel Bacillus strain with Clostridium tyrobutyricum ATCC 25755 to produce butyric acid from sucrose

Background: Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755(T); however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755(T) to ferment sucrose to butyric acid. Results: B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755(T) to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755(T) cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37 degrees C. Under these conditions, the final butyrate concentration was 34.2 +/- 1.8 g/L with yields of 0.35 +/- 0.03 g (butyrate)/g (sucrose) and maximum productivity of 0.3 +/- 0.04 g/L/h. Conclusions: Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated.open2

Optochemical control of gene expression by photocaged guanine and riboswitches

Optical control of biomolecules via engineered proteins or photoactive small molecules has had a profound impact on biology. However, optochemical tools to control RNA functions in living cells are relatively limited. We synthesized a photoactivatable (photocaged) guanine to modulate gene expression under riboswitch control in both mammalian cells and Escherichia coli by light

Outer Membrane Porin F in E. coli Is Critical for Effective Predation by Bdellovibrio

Bdellovibrio and like organisms (BALOs) are a unique bacterial group that live by predating on other bacteria, consuming them from within to grow and replicate before the progeny come out to complete the life cycle. The mechanisms by which these predators recognize their prey and differentiate them from nonprey bacteria, however, are still not clear. Through genetic knockout and complementation studies in different Escherichia coli strains, we found that Bdellovibrio bacteriovorus strain 109J recognizes outer membrane porin F (OmpF) on the E. coli surface and that the activity of the E. coli EnvZ-OmpR regulatory system significantly impacts predation kinetics. OmpF is not the only signal by which BALOs recognize their prey, however, as B. bacteriovorus could eventually predate on the E. coli ??ompF mutant after prolonged incubation. Furthermore, recognizing OmpF as a prey surface structure was dependent on the prey strain, as knocking out OmpF protein homologues in other prey species, including Escherichia fergusonii, Klebsiella pneumoniae, and Salmonella enterica, did not always reduce the predation rate. Consequently, although OmpF was found to be an important surface component used by Bdellovibrio to efficiently recognize and attack E. coli, future work is needed to determine what other prey surface structures are recognized by these predators

Study on the Effect of the addition of Synthesized Nano scale Lead Oxide for Concrete Samples used in Gamma-Ray Shielding

Our goal in this research is to investigate the effect ofconcrete incorporated with nanoparticles of lead oxide inpowder form by different percentages on Gamma-ray shieldingcharacteristics. The lead oxide nanostructure was synthesizedthrough the reaction of citric acid (C6H7O8.H2O) solution andlead acetate (Pb (C2H3O2)) solution as stabilizer and precursor,respectively. In this synthesis, the prepared lead oxidenanostructure was characterized by X-Ray Diffraction (XRD)and Scanning Electron Microscopy (SEM) techniques. Theprepared PbO consists of the crystallites about 50 nm. Theconcrete samples were prepared according to the localstandards of building materials and doped with PbO nanopowders by different percentages, 0%, 5%and 10% (by weight)by replacing cement and keeping constant w/c ratio. Moreover,commercial PbO bulk powder additive was used to check theeffect of particle size on concrete attenuation properties. The γ-rays attenuation coefficients were measured as a function of theadditive percentage of nanoparticles of lead oxides and using γ-ray point source,126Ra with different energies in the range(0.295- 1.73) MeV. The results were compared with that fornormal concrete incorporated with the same percentage of bulklead oxide. It was found that the γ-ray attenuation coefficientfor concrete doped with nanoparticles of PbO was slightlyimproved

Predatory Bacteria: A Potential Ally against Multidrug-Resistant Gram-Negative Pathogens

Multidrug-resistant (MDR) Gram-negative bacteria have emerged as a serious threat to human and animal health. Bdellovibrio spp. and Micavibrio spp. are Gram-negative bacteria that prey on other Gram-negative bacteria. In this study, the ability of Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus to prey on MDR Gram-negative clinical strains was examined. Although the potential use of predatory bacteria to attack MDR pathogens has been suggested, the data supporting these claims is lacking. By conducting predation experiments we have established that predatory bacteria have the capacity to attack clinical strains of a variety of ß-lactamase-producing, MDR Gram-negative bacteria. Our observations indicate that predatory bacteria maintained their ability to prey on MDR bacteria regardless of their antimicrobial resistance, hence, might be used as therapeutic agents where other antimicrobial drugs fail. © 2013 Kadouri et al