The effect of the electric field on lag phase, β-galactosidase production and plasmid stability of a recombinant Saccharomyces cerevisiae strain growing on lactose

Ethanol and β-galactosidase production from cheese whey may significantly contribute to minimise environmental problems while producing value from lowcost raw materials. In this work, the recombinant Saccharomyces cerevisiae NCYC869-A3/pVK1.1 flocculent strain expressing the lacA gene (coding for β-galactosidase) of Aspergillus niger under ADHI promoter and terminator was used. This strain shows high ethanol and β-galactosidase productivities when grown on lactose. Batch cultures were performed using SSlactose medium with 50 gL−1 lactose in a 2-L bioreactor under aerobic and microaerophilic conditions. Temperature was maintained at 30 °C and pH 4.0. In order to determine the effect of an electric field in the fermentation profile, titanium electrodes were placed inside the bioreactor and different electric field values (from 0.5 to 2 Vcm−1) were applied. For all experiments, β-galactosidase activity, biomass, protein, lactose, glucose, galactose and ethanol concentrations were measured. Finally, lag phase duration and specific growth rate were calculated. Significant changes in lag phase duration and biomass yield were found when using 2 Vcm−1. Results show that the electric field enhances the early stages of fermentation kinetics, thus indicating that its application may improve industrial fermentations’ productivity. The increase in electric field intensity led to plasmid instability thus decreasing β-galactosidase production.The authors gratefully acknowledge Fundacao para a Ciencia e a Tecnologia (Portugal) for the scholarships SFRH/BD/11230/2002 and SFRH/BDP/63831/2009 granted to authors I. Castro and C. Oliveira, respectively

FLPe functions in zebrafish embryos

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0–4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C

Fragilities Caused by Dosage Imbalance in Regulation of the Budding Yeast Cell Cycle

Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1∶1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial “genetic tug-of-war” experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems