http://www.biomedcentral.com/bmcbiotechnol/ The editor's pick of recent articles published by BMC Biotechnology 2012-05-03T00:00:00Z Background: The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their "brightness" and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results: The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions: From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here. http://www.biomedcentral.com/1472-6750/12/17 Davd GJ Mann Laura L Abercrombie Mary R Rudis Reginald J Millwood John R Dunlap C Neal Stewart BMC Biotechnology 2012, 12:17 2012-05-03T00:00:00Z 10.1186/1472-6750-12-17 Brightest reporter in transgenic plants The tandem dimer tdTomato and the monomeric mOrange are the two highest fluorescing orange fluorescent proteins (OFPs) available as reporters for transgenic plants, when targeted to the endoplasmic reticulum. /content/figures/1472-6750-12-17-toc.gif BMC Biotechnology 1472-6750 12 17 2012-05-03T00:00:00Z PDF Background: There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Results: Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions: LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations. http://www.biomedcentral.com/1472-6750/12/15 Guy Kiddle Patrick Hardinge Neil Buttigieg Olga Gandelman Clint Pereira Cathal J McElgunn Manuela Rizzoli Rebecca Jackson Nigel Appleton Cathy Moore Laurence C Tisi James AH Murray BMC Biotechnology 2012, 12:15 2012-04-30T00:00:00Z 10.1186/1472-6750-12-15 Novel technique for GMO detection Genetically modified maize DNA can be effectively detected even at low levels of contamination by the coupling of loop mediated isothermal amplification (LAMP) to a bioluminescent output produced in real-time (BART). /content/figures/1472-6750-12-15-toc.gif BMC Biotechnology 1472-6750 12 15 2012-04-30T00:00:00Z PDF Background: Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation. Results: The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes. Conclusions: Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method. http://www.biomedcentral.com/1472-6750/12/11 Caterina Boniello Torsten Mayr Juan M Bolivar Bernd Nidetzky BMC Biotechnology 2012, 12:11 2012-03-28T00:00:00Z 10.1186/1472-6750-12-11 Monitoring intra-particle pH A Dual-Lifetime Referencing (DLR) method based on the luminophores fluorescein and Ru(dpp) allows the measurement of pH within the particles of carrier-bound immobilized enzymes and has potential as an analytical tool in biocatalysis. /content/figures/1472-6750-12-11-toc.gif BMC Biotechnology 1472-6750 12 11 2012-03-28T00:00:00Z XML Translational medicine depends on a rapid and efficient exchange of results between the bench and the bedside. A recent example from the field of cancer immunotherapy highlights the essential nature of this exchange. Methods have been developed to convert a patient's cytotoxic T cells into efficient and specific killers of cancer cells in patients with leukemia. By using recombinant DNA techniques, a lentiviral vector was constructed to express chimeric antigen receptors in cytotoxic T cells from patients with advanced chronic lymphocytic leukemia. The purpose of the chimeric receptors was to direct the cytotoxic T cell activity against cells causing the cancer. The effect of infusing the engineered T cells back into the cancer patients was tested in a Phase I trial at the University of Pennsylvania, and the initial results were described in two articles from the research team of Dr. Carl June. The remarkable success of this trial should energize further applications of biotechnology in the development of new cancer immunotherapies. http://www.biomedcentral.com/1472-6750/12/6 Marko Radic BMC Biotechnology 2012, 12:6 2012-02-08T00:00:00Z 10.1186/1472-6750-12-6 Engineered T cells against leukemia Marko Radic discusses recent advances in cancer immunotherapy, whereby cytotoxic T cells from patients with leukemia are turned into efficient and specific killers of their own cancer cells. /content/figures/1472-6750-12-6-toc.gif BMC Biotechnology 1472-6750 12 6 2012-02-08T00:00:00Z XML Background: Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. Results: We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans. Conclusion: Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations. http://www.biomedcentral.com/1472-6750/12/1 Eriko Kage-Nakadai Hiroyuki Kobuna Osamu Funatsu Muneyoshi Otori Keiko Gengyo-Ando Sawako Yoshina Sayaka Hori Shohei Mitani BMC Biotechnology 2012, 12:1 2012-01-05T00:00:00Z 10.1186/1472-6750-12-1 UV/TMP method for C. elegans manipulation A method utilizing ultraviolet trimethylpsoralen (UV/TMP) based on familiar extrachromosomal transgenics offers an alternative to Mos1 transposon or microparticle bombardment in the creation of single- or low-copy chromosomal integrated lines of Caenorhabditis elegans. /content/figures/1472-6750-12-1-toc.gif BMC Biotechnology 1472-6750 12 1 2012-01-05T00:00:00Z XML Background: Water contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies. Results: A gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure. Conclusions: EVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples. http://www.biomedcentral.com/1472-6750/11/123 Takahiro Imai Daisuke Sano Takayuki Miura Satoshi Okabe Keishi Wada Yoshifumi Masago Tatsuo Omura BMC Biotechnology 2011, 11:123 2011-12-16T00:00:00Z 10.1186/1472-6750-11-123 Bacterial protein detects enteric viruses A broad spectrum virus-binding protein derived from the bacterial chaperon protein GroEL and able to bind rotavirus, norovirus and poliovirus, could be used to detect diverse enteric viruses simultaneously in water and fecal samples. /content/figures/1472-6750-11-123-toc.gif BMC Biotechnology 1472-6750 11 123 2011-12-16T00:00:00Z XML