text:
A green-light inducible lytic system for cyanobacterial cells
Kotone Miyake1, 2,
Koichi Abe1, 2,
Stefano Ferri1, 2,
Mitsuharu Nakajima1, 2,
Mayumi Nakamura1, 2,
Wataru Yoshida1, 2,
Katsuhiro Kojima1, 2,
Kazunori Ikebukuro1, 2 and
Koji Sode1, 2Email author
Biotechnology for Biofuels20147:56
© Miyake et al.; licensee BioMed Central Ltd. 2014
Published: 9 April 2014
Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds.
We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light.
These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments.
A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort and energy, due to the fragile nature of the induced cells. Furthermore, the use of light-sensing two-component systems to regulate the expression of exogenous genes in cyanobacteria promises to replace conventional chemical inducers in many bioprocess applications, impacting the limiting water management issues.
Self-lysis
Two-component system
T4 bacteriophage
There is an increasingly urgent search for sustainable and renewable energy alternatives to fossil fuels [1]. Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land, which does not compete with terrestrial agricultural crops for land or water [2]. Furthermore, many different synthetic biology tools are now available for the convenient genetic engineering of cyanobacteria [3–7]. Although many strategies are now being reported for producing valuable compounds in genetically engineered cyanobacteria, an efficient method of collecting the synthesized compounds remains to be developed. Among the various biofuel-related products expected to be produced in cyanobacteria, there is a particular focus on the accumulation of intracellular lipids [8]. A successful downstream process to recover intracellular lipids will require disrupting the native structure of the cyanobacterial cell prior to extraction with solvents. Mechanical methods have generally been used to disrupt cells, such as by cell homogenizer, bead mill, ultrasound, French press, and autoclave. Non-mechanical methods, such as freeze-thaw, organic solvents, detergents, osmotic shock, acid, base, and enzyme reactions have also been employed. The use of supercritical methanol to convert wet algae to biodiesel has also been recently reported [9]. However, energy consumption is a concern for mechanical methods, while non-mechanical methods require additional steps for removing the chemicals, resulting in extra costs. More efficient and cost-effective methods must therefore be designed.
An additional advantage of using algae for biofuel production is the possibility of using water that is unsuitable for land crops, such as saline water from aquifers and seawater, especially when using marine-derived strains. At the same time, strict water management is one of the greatest issues in biofuel production. Considering the use of huge quantities of water required for algal cultivation, water recycling is an essential feature. Most of the genetic tools developed for cyanobacterial bioprocesses are based on chemical induction. Since the removal of such chemicals is required for recycling the water for cultivation, chemical induction methods are not appropriate for industrial processes.
Liu and Curtiss reported pioneer work on a programmed cell lysis system for the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) [10]. This system consists of a constitutively expressed P22 bacteriophage-derived endolysin and auxiliary enzyme, and P22 holin expressed under a nickel-ion inducible promoter, which originates from the Synechocystis nrsBACD operon involved in Ni2+ resistance [11]. This lysis system was somewhat successful in that the expression of lysis genes could be regulated inside cyanobacterial cells, and therefore contributes to reducing the energy required for extraction of the intracellular product. However, the use of a nickel-ion induction system limits its feasible application for future biofuel production processes [12].
We propose a novel process to prepare lytic cyanobacteria that can be regulated by a physical signal, green-light illumination, for future use in the recovery of biofuel-related compounds (Figure 1). Synechocystis possesses light responsive two-component systems, such as the CcaS/CcaR two-component system, which responds to red/green light [13]. The cyanobacteriochrome CcaS and response regulator CcaR regulate the expression of cpcG2, encoding a phycobilisome linker protein. The expression of cpcG2 is up-regulated by green-orange light (550-600 nm) and down-regulated by red light (672 nm). By placing T4 phage-derived lysis genes under the control of the cpcG2 promoter on a broad host range vector and introducing it into Synechocystis, we designed a green-light inducible lytic cyanobacteria for future use in the recovery of biofuel-related compounds.
Regulation of cyanobacterial lytic system by green light. Schematic diagram of a novel system to prepare lytic cyanobacterial cells by physical signal, green-light illumination. The lysis system is composed of the CcaS/CcaR green-light regulated two-component system derived from Synechocystis, combined with T4 phage-derived lysis genes under the CcaS/CcaR-regulated cpcG2 promoter. CcaS is a sensor histidine kinase that activates its cognate response regulator, CcaR, under green light to induce the expression of the cpcG2 gene. Under red light, CcaS is in an 'off-state' and expression of the target gene is not activated (A). However under green light, CcaS is in an 'on-state', resulting in activation of CcaR by phosphorylation and a concomitant induction of the target gene expression (B). Holin forms a 'tunnel' in the plasma membrane, which provides endolysin access to the peptidoglycan and allows it to enzymatically break it down, leading to lysis. Antiholin, which is regulated by a weak constitutive promoter, blocks holin and prevents premature lysis due to low background levels of endolysin.
Green-light induction of cell lysis
The T4 phage-derived lysis genes encoding holin, endolysin, and antiholin [14–17] were introduced into Synechocystis on the broad host range vector pKT230 [18], which has been utilized for the recombinant expression in both freshwater and marine cyanobacterial strains [19–24]. To link the lysis genes to the endogenous CcaS/CcaR two-component system for green-light regulation, the region upstream of the Synechocystis cpcG2 gene was amplified by PCR and inserted upstream of the T4 holin and endolysin genes (Figure 1). Antiholin was inserted downstream of a weak constitutive promoter to prevent premature lysis from leaky expression of the holin and endolysin genes prior to induction (data not shown). Synechocystis transformed with the pKT230 empty vector (Synechocystis/pKT230) or pKT230 harboring the above lysis cassette (Synechocystis/pKTLYS) were grown at 30°C under red-light illumination (660 nm, 20 μmol m-2 s-1) alone or under simultaneous red- and green-light illumination (520 nm, 20 μmol m-2 s-1).
Figure 2 shows the cell growth of Synechocystis/pKT230 (Figure 2A, B) and Synechocystis/pKTLYS (Figure 2C, D) under continuous red-light illumination (Figure 2A, C) or under red-light illumination for 84 hours followed by simultaneous red- and green-light illumination (Figure 2B, D). In order to investigate cell lysis, phycocyanin-derived fluorescence of the culture supernatant was also monitored as an indicator of the leakage of intracellular components [25]. Cell growth reached the early logarithmic phase after approximately 80 hours of cultivation under red-light illumination, with no significant difference in growth rate between Synechocystis/pKT230 and Synechocystis/pKTLYS. In both cultures, no phycocyanin-derived fluorescence was observed under red-light illumination. After 84 hours of incubation under red-light illumination, some cells were transferred for a further 64 hours under simultaneous red- and green-light illumination (Figure 2B, D). The additional green-light illumination did not cause any significant change in the cellular growth of Synechocystis/pKT230 (Figure 2B) when compared with cultivation under red-light illumination alone (Figure 2A). In contrast, the culture of Synechocystis/pKTLYS under simultaneous red- and green-light illumination showed a considerable decrease in growth rate at about 48 hours after starting green-light illumination (Figure 2D). The cell growth of Synechocystis/pKTLYS under only red-light illumination (Figure 2C) was similar with that of the Synechocystis/pKT230 cultures (Figure 2A, B). More remarkably, the drastic increase in the phycocyanin fluorescence in the supernatant was observed only in Synechocystis/pKTLYS under simultaneous red- and green-light illumination, at about 64 hours after starting green-light illumination. Considering that no phycocyanin-derived fluorescence was observed from the culture supernatant of Synechocystis/pKT230 under both illumination conditions (Figure 2A, B), as well as from the culture supernatant of Synechocystis/pKTLYS under only red-light illumination (Figure 2C), the observed fluorescence increase from green-light illuminated Synechocystis/pKTLYS was due to the induction of lysis genes by the introduced functional green-light regulated promoter region, causing the partial leakage of intracellular proteins including phycocyanin. The observed decrease in the cell growth of green-light illuminated Synechocystis/pKTLYS culture was possibly due to cell death caused by increased cell permeability resulting from the green-light induced lysis gene products.
Growth curves and supernatant fluorescence of Synechocystis cultures. Growth curves (circles with solid line) and supernatant fluorescence intensity (squares with dashed line; excitation: 620 nm, emission: 650 nm), representing leaked phycocyanin, of Synechocystis/pKT230 (A and B) and Synechocystis/pKTLYS (C and D). The cells were grown under either continuous red-light illumination (A and C) or under red light for 84 hours followed by simultaneous red- and green-light illumination (B and D).
To confirm that the green-light sensing system induced the lysis genes, we investigated the mRNA level of T4 holin, whose transcription was designed to be regulated by the green-light activated response regulator CcaR. The culture of Synechocystis/pKTLYS was sampled at 2, 5, 8, 12, and 24 hours after starting green-light illumination, and the level of T4 holin mRNA was analyzed by reverse transcription PCR (RT-PCR) (Figure 3). The transcriptional level of T4 holin after initiating green-light illumination was approximately threefold higher than those of cells cultivated under only red-light illumination. After 24 hours, the transcription level under green light decreased down to the basal mRNA level observed under red light. The T4 holin gene was therefore regulated under the green-light sensing system, and was induced by green-light illumination prior to the observable phenotypic changes, such as the leakage of phycocyanin and decrease of cell growth.
Analysis of T4 holin transcription level by RT-PCR. Total RNA was purified from Synechocystis/pKTLYS at 2, 5, 8, 12, and 24 hours after green-light induction to analyze the relative transcription level of the holin gene under red light (black bars) and green light (white bars).
These results indicated that our constructed green-light induced lytic system was repressed under the cultivation conditions with red-light illumination, and was clearly induced under green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant.
Characterization of lytic cyanobacterial cells
The prepared lytic cells of Synechocystis/pKTLYS expressing lysis genes under green-light illumination were then characterized by monitoring leakage of phycocyanin under various treatments to confirm their increased cell fragility, such as detergent (Triton X-100), freeze-thaw, or osmotic shock treatments (Figure 4). The condition of the Synechocystis plasma membrane was also investigated by SYTOX Green nucleic acid staining (Life technologies, MD, United States) (Figure 5).
Characterization of lytic cyanobacterial cells under different stress treatments. Synechocystis/pKT230 (pKT230) and Synechocystis/pKTLYS (pKTLYS) were incubated for 64 hours under only red light (black bars) or under simultaneous red and green light (white bars). Cells were then subjected to freeze-thaw, osmotic shock, or Triton X-100 treatments, followed by measurement of the supernatant fluorescence intensity, representing the released phycocyanin.
Microscopic images of SYTOX green-stained Synechocystis . Synechocystis/pKT230 grown under continuous red light (A and B) or under red and green light (C and D), and Synechocystis/pKTLYS grown under red light (E and F) or under red and green light (G and H) were observed by visible-light microscopy (A, C, E, and G) and by fluorescence microscopy (B, D, F, and H). All cells were grown for 64 hours and then incubated with 5 μM SYTOX Green nucleic acid stain for five minutes at room temperature. Fluorescence microscopy was performed on a Biozero BZ- 8000 (Keyence, Osaka, Japan) (excitation: 485 nm, emission: 520 nm).
Triton X-100 treatment did not cause significant leakage of phycocyanin from Synechocystis/pKT230 cells to the supernatant. However, Triton X-100 treatment caused cells from Synechocystis/pKTLYS induced under green light to release over 20-fold more phycocyanin than Synechocystis/pKTLYS cultivated under continuous red light. Similarly, osmotic-shock and freeze-thaw treatments resulted in 1.2 and 11%, respectively, of phycocyanin release to the supernatant from Synechocystis/pKTLYS cultivated under continuous red-light illumination compared to red and green light.
SYTOX Green is a fluorescent indicator dye commonly used to assess cell death by tightly binding to nucleic acids in cells whose plasma membranes have lost integrity [26]. Microscopic analysis revealed that only 2.7 ± 1.7% and 3.3 ± 3.3% of the Synechocystis/pKT230 cells cultivated under continuous red-light illumination and under green-light illumination, respectively, were stained with SYTOX Green. Comparable results were observed with the non-induced Synechocystis/pKTLYS, where only 5.7 ± 2.3% of the cells were stained. In sharp contrast, 39 ± 19% of the Synechocystis/pKTLYS cells cultivated under green light were stained, indicating that a considerable fraction of the cells had compromised membranes as a result of the green-light induced lysis genes.
These results demonstrate that the green-light induced lytic system caused partial-cell lysis, rendering the cell membranes fragile. These cells could then easily be further disrupted under mild stress, such as osmotic shock or incubation with detergent. Thus, prepared lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells, since the fragile nature of the induced cells helps to minimize the energy needed to disrupt the cell wall. Moreover, the fact that the developed system is induced by a signal transduction system responding to a physical signal (a change in the wavelength of light) is definitely advantageous over chemical induction, which results in the culture medium remaining contaminated by the chemical inducer. One of the important issues to be addressed before realizing the microalgal bioprocesses for biofuels is the minimization of water used for cultivation; the recycling of the culture medium is therefore ideal. However, compounds used in chemical-based signal transduction systems are technically quite difficult to remove or recover, and are also energetically unfavorable for industrial applications. In practice, cyanobacterial cells will be cultured under continuous illumination of sunlight with a filter to cut out green light, and will then be easily induced by removing the filter, allowing all wavelengths of light, including green light, to reach the cyanobacterial cells.
Another potential beneficial feature of the green-light inducible lytic system is that cell growth slows down greatly. The SYTOX Green staining experiment indicated that the system can be used to induce cell death by green-light exposure. This property suggests a future possibility to construct a photosynthetic genetically modified organism (GMO) that is unable to survive under sunlight exposure. Although the system still needs to be improved, we have demonstrated a potential future platform concept for photoautotrophic GMOs unable to survive if accidentally released into the natural habitat.
We succeeded in genetically engineering a green-light inducible lytic system in Synechocystis. Illumination of the engineered cyanobacteria resulted in a considerably reduced growth rate and we demonstrated using osmotic shock, freeze-thaw, and detergent treatments, as well as by SYTOX Green staining, that the plasma membranes were compromised. The use of light-sensing two-component systems to regulate the expression of exogenous genes in cyanobacteria promises to replace conventional chemical inducers in many bioprocess applications, impacting the limiting water management issues. The ability to use such a system to regulate cyanobacterial lysis also has the potential to replace the inconvenient and expensive mechanical cell disruption methods. The proposed system demonstrated a potential future platform concept for photoautotrophic GMOs that are unable to survive under sunlight if accidentally released in the natural habitat.
Plasmids and strains
All gene constructs were created by standard DNA manipulations using Escherichia coli DH5α. Using overlap extension PCR, we constructed the green-light inducible lysis cassette containing the following in order: (1) PcpcG2, the region containing the promoter regulated by CcaS-CcaR green-light sensor; (2) T4 holin gene; (3) T4 endolysin gene; (4) double transcriptional terminator (BioBrick part BBa_B0015) (BioBrick foundation, MA, United State); (5) constitutive promoter (BioBrick part BBa_J23116); (6) antiholin gene; and (7) double transcriptional terminator (BioBrick part BBa_B0015). To increase the expression level of the T4 holin gene, the exogenous Shine-Dalgarno-like sequence from the upstream region of the cpcB gene of Synechococcus sp. PCC 7002 (5′-TAGGAGATAAAAATA-3′) was inserted upstream of the start codon of the T4 holin gene by inverse PCR [27]. The lysis cassette (entire sequence and gene organization provided in Additional files 1 and 2) was inserted into the Pst I sites of the RSF1010-derived broad host range vector pKT230, removing the kanamycin resistance gene and keeping the streptomycin resistance gene. All primers used for constructing the lysis cassette are listed in Additional file 3. Synechocystis was grown with gentle shaking (100 rpm) in 100 mL Erlenmeyer flasks containing 40 mL BG11 medium (0% NaCl) [28] supplemented with 10 mM TES–KOH (pH 7.4). Synechocystis transformed with pKT230-derived vectors were grown in the same medium supplemented with 50 μg/mL streptomycin. Cyanobacterial cells were cultured at 30°C under continuous overhead illumination provided by red-light (620 nm) light-emitting diode (Panasonic, Osaka, Japan) at an intensity of 20 μmol m-2 s-1. For gene induction, the cultures were also illuminated from below by green-light (520 nm) light-emitting diode (NK System, Osaka, Japan) at an intensity of 20 μmol m-2 s-1.
Transformation of Synechocystis
A 40 mL linear phase culture (Optical density at 730 nm (OD730) = 0.4) of Synechocystis was harvested by centrifugation. The pellet was washed three times with decreasing volumes (30 mL, 20 mL, and 15 mL) of 1 mM HEPES buffer and re-suspended in 200 μL of the remaining liquid. For electroporation, 40 μL of the suspended cells were mixed with 500 ng plasmid DNA inside a 2 mm cuvette and pulsed with a Gene Pulser Xcell (Biorad, CA, United States) at 12 kV/cm at a time constant of 5 ms. Immediately following the pulse, 1 mL of BG11 was added into the cuvette and transferred to 5 mL BG11 in an Erlenmeyer flask to undergo a one-day recovery at 30°C under red-light illumination (660 nm, 20 μmol m-2 s-1). The cells were then concentrated by centrifugation to 500 μL, mixed with 5 mL melted BG11 soft agar, and spread onto a BG11 plate containing 50 μg/mL streptomycin). The plate was incubated at 30°C under red light and transformed colonies were confirmed by PCR.
Lysis evaluation in Synechocystis
Synechocystis transformed with the pKT230 empty vector or pKT230 harboring the lysis cassette, respectively Synechocystis/pKT230 and Synechocystis/pKTLYS, were inoculated into 40 mL BG11 liquid medium at OD730 = 0.1 and grown under red light as described above. When the transformants reached exponential growth, at OD730 near 0.5, half the cultures were kept under red light and the other half were transferred to simultaneous red- and green-light illumination, as described above. The samples were then monitored for two days by measuring the OD730 of the cultures and the fluorescence intensity (excitation: 620 nm, emission: 650 nm) of the supernatant after centrifugation (4,000 × g, 2 min.) using a plate reader (Thermo Fisher Scientific Inc., MA, United States). All conditions were investigated in triplicate.
RNA extraction and transcription analysis of holin by RT-PCR
Total RNA was isolated from 25 mL of culture using NucleoSpin RNA Clean-up kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions and treated with DNase to remove genomic DNA. The extracted RNA was reverse transcribed to cDNA using PrimeScript® RT reagent kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). The transcription levels of 16S rRNA and T4 holin were analyzed by preparing reaction solutions with SYBR Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan) using primers shown in Additional file 3. The transcription level of T4 holin was normalized by the transcription level of 16S rRNA based on the ∆∆Ct method.
Cell fragility assay
To evaluate whether the cells expressing lysis genes were rendered fragile, the effects of osmotic shock, freeze-thaw, and detergent on cell integrity were investigated. After 64 hours of green-light induction, 1 mL of green-light illuminated or green- and red-light illuminated Synechocystis/pKT230 and Synechocystis/pKTLYS were harvested by centrifugation. The osmotic-shock treatment was carried out by re-suspending the harvested pellet in 1 mL deionized water and incubating 1 hour at room temperature. The effect of freeze-thaw was investigated by placing the harvested pellet in –80°C for 30 minutes followed by 30°C for 20 minutes. This freeze-thaw was repeated once and the pellet was then re-suspended in fresh BG11 medium. The detergent treatment was carried out by re-suspending the harvested pellet in BG11 containing 1% Triton X-100 and incubating for 1 hour at room temperature. All treated samples were then centrifuged (4,000 × g, 2 min) and the fluorescence intensity (excitation: 620 nm, emission: 650 nm) of the supernatant measured.
GMO:
Optical density
RT-PCR:
Reverse transcription PCR
Synechocystis:
Synechocystis sp. PCC 6803.
This work was supported financially by the Core Research of Evolutional Science & Technology program (CREST) from the Japan Science and Technology Agency (JST).
13068_2013_453_MOESM1_ESM.png Additional file 1:The cyanobacterial lysis device pKTLYS (bottom) was created by inserting lysis genes into the broad-host-range vector pKT230 (top). The promoter region of the Synechocystis cpcG2 gene (recognized by the CcaR response regulator) was inserted upstream of the T4 holin and T4 endolysin genes. T4 antiholin is constitutively expressed under a weak constitutive promoter (BioBrick BBa_J23116). The cassette containing the above lysis genes was inserted into the PstI sites of pKT230 to create pKTLYS. (PNG 192 KB)
13068_2013_453_MOESM2_ESM.docx Additional file 2:Sequence of T4 lysis cassette that was inserted into the PstI sites of pKT230 to create pKTLYS, used in this study. The cpcG2 promoter region (green) and an SD-like sequence (red) of the Synechococcus sp. PCC 7002 cpcB gene were inserted upstream of the T4 holin (blue) and T4 endolysin (orange) genes. T4 antiholin (pink) is constitutively expressed. (DOCX 228 KB)
13068_2013_453_MOESM3_ESM.docx Additional file 3:List of primers used in this work.[13]. (DOCX 92 KB)
13068_2013_453_MOESM8_ESM.tif Authors' original file for figure 5
KM performed the evaluation of lysis systems, KA constructed the vectors used in this work, MNakamura evaluated the effect of introducing SD-like sequence in front of ATG for green-light induction. MNakajima, SF, KK, KI, and KS performed data analysis. KM, WY, KA, SF, KI, and KS participated in its design and coordination and wrote the manuscript. All authors drafted and revised the work. All authors read and approved the final manuscript.
Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
JST, CREST, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
Zhou J, Li Y: Engineering cyanobacteria for fuels and chemical production. Protein Cell 2010, 1: 207-210. 10.1007/s13238-010-0043-9View ArticleGoogle Scholar
Ducat DC, Way JC, Silver PA: Engineering cyanobacteria to generate high-value products. Trends in Biotechnol 2011, 29: 95-103. 10.1016/j.tibtech.2010.12.003View ArticleGoogle Scholar
Huang HH, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a synthetic biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38: 2577-2593. 10.1093/nar/gkq164View ArticleGoogle Scholar
Wang B, Wang J, Zhang W, Meldrum DR: Application of synthetic biology in cyanobacteria and algae. Front Microbiol 2012, 3: 1-15.Google Scholar
Niederholtmeyer H, Wolfstädter BT, Savage DF, Silver PA, Way JC: Engineering cyanobacteria to synthesize and export hydrophilic products. Appl Environ Microbiol 2010, 76: 3462-3466. 10.1128/AEM.00202-10View ArticleGoogle Scholar
Deng MD, Coleman JR: Ethanol synthesis by genetic engineering in cyanobacteria. Appl Environ Microbiol 1999, 65: 523-528.Google Scholar
Oliver JW, Machado IM, Yoneda H, Atsumi S: Cyanobacterial conversion of carbon dioxide to 2, 3-butanediol. Proc Natl Acad Sci U S A 2013, 110: 1249-1254. 10.1073/pnas.1213024110View ArticleGoogle Scholar
Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A: Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 2008, 54: 621-639. 10.1111/j.1365-313X.2008.03492.xView ArticleGoogle Scholar
Patil PD, Gude VG, Mannarswamy A, Deng S, Cooke P, Munson-McGee S, Rhodes I, Lammers P, Nirmalakhandan N: Optimization of direct conversion of wet algae to biodiesel under supercritical methanol conditions. Bioresour Technol 2011, 102: 118-122. 10.1016/j.biortech.2010.06.031View ArticleGoogle Scholar
Liu X, Curtiss R 3rd: Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc Natl Acad Sci U S A 2009, 106: 21550-21554. 10.1073/pnas.0911953106View ArticleGoogle Scholar
López-Maury L, García-Domínguez M, Florencio FJ, Reyes JC: A two-component signal transduction system involved in nickel sensing in the cyanobacterium Synechocystis sp. PCC 6803. Mol Microbiol 2002, 43: 247-256. 10.1046/j.1365-2958.2002.02741.xView ArticleGoogle Scholar
Singh J, Gu S: Commercialization potential of microalgae for biofuels production. Renew Sust Energ Rev 2010, 14: 2596-2610. 10.1016/j.rser.2010.06.014View ArticleGoogle Scholar
Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M: Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A 2008, 105: 9528-9533. 10.1073/pnas.0801826105View ArticleGoogle Scholar
Young R, Wang I, Roof WD: Phages will out: strategies of host cell lysis. Trends Microbiol 2000, 8: 120-128. 10.1016/S0966-842X(00)01705-4View ArticleGoogle Scholar
Gründling A, Manson MD, Young R: Holins kill without warning. Proc Natl Acad Sci U S A 2001, 98: 9348-9352. 10.1073/pnas.151247598View ArticleGoogle Scholar
Tran TAT, Struck DK, Young R: Periplasmic domains define holin-antiholin interactions in T4 lysis inhibition. J Bacteriol 2005, 187: 6631-6640. 10.1128/JB.187.19.6631-6640.2005View ArticleGoogle Scholar
Tran TA, Struck DK, Young R: The T4 RI antiholin has an N-terminal signal anchor release that targets it for degradation by DegP. J Bacteriol 2007, 189: 7618-7625. 10.1128/JB.00854-07View ArticleGoogle Scholar
Bagdasarian M, Lurz R, Rückers B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN: Specific-purpose plasmid cloning vectors: II broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas . Gene 1981, 16: 237-247. 10.1016/0378-1119(81)90080-9View ArticleGoogle Scholar
Sode K, Tatara M, Takeyama H, Burgess JG, Matsunaga T: Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp. Appl Microbiol Biotechnol 1992, 37: 369-373. 10.1007/BF00210994View ArticleGoogle Scholar
Sode K, Tatara M, Matsunaga T: Maintenance of broad host range vector pKT230 in marine unicellular cyanobacteria. FEMS Microbiol Lett 1992, 99: 73-78. 10.1111/j.1574-6968.1992.tb05544.xView ArticleGoogle Scholar
Sode K, Tatara M, Hatano N, Matsunaga T: Foreign gene expression in marine cyanobacteria under pseudo-continuous culture. J Biotechnol 1994, 33: 243-248. 10.1016/0168-1656(94)90072-8View ArticleGoogle Scholar
Sode K, Hatano N, Tatara M: Cloning of marine cyanobacterial promoter for foreign gene expression using a promoter probe vector. Appl Biochem Biotechnol 1996, 59: 349-360. 10.1007/BF02783576View ArticleGoogle Scholar
Sode K, Hayashi T, Tatara M, Hatano N, Yoshida H, Takeyama H, Matsunaga T, Oshiro T: Recovery of marine cyanobacterial recombinant product using fish feed organisms. J Marine Biotechnol 1996, 4: 82-86.Google Scholar
Sode K, Yamamoto Y, Hatano N: Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene. J Marine Biotechnol 1998, 6: 174-177.Google Scholar
Aaron JT, Yuping AC, Alexander NG: Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host. Proc Natl Acad Sci U S A 2001, 98: 10560-10565. 10.1073/pnas.181340998View ArticleGoogle Scholar
Roth BL, Poot M, Stephen YT, Millard PJ: Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 1997, 63: 2421-2431.Google Scholar
Abe K, Miyake K, Nakamura M, Kojima K, Ferri S, Ikebukuro K, Sode K: Engineering of a greenlight inducible gene expression system in Synechocystis sp. PCC6803. Microbial Biotechnol 2014, 7: 177-183. 10.1111/1751-7915.12098View ArticleGoogle Scholar
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G: Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 1971, 35: 171-205.Google Scholar


Dissolve starch with 1 glass of cold water and stir well to avoid lumps. Pour sugar into the saucepan with the remaining water, stirring, bring to the boil and cook over medium heat for 15–20 minutes.
When the sugar syrup begins to boil strongly, pour in the starch solution, reduce the heat, put finely grated zest and, stirring constantly, boil until the mass thickens.
When the Turkish delight lags behind the walls of the dishes, add citrus oil, mix again and put the mass on a baking sheet filled with parchment paper. Align Turkish delight with a spoon moistened in cold water and let it cool for 4–5 hours. Slice Turkish delight into small cubes, roll in powdered sugar.


The Mediterranean Sea meets the Taurus Mountains with a breathtaking view, top of the cliffs, 156 rooms with panoramic views of the sea intertwined with the city and the unique beauty of blue and green, we will be your constant address in your holiday and business trips.
The unique location of our hotel puts it in a special place for those who want to explore Antalya, as well as for meeting and congress for travelers.
95 total rooms includes two single beds with sea view. In all of our rooms with a total area of 17 m2; Coffee Table and Chair, Telephone, Central Air Conditioning System, Satellite TV, Mini bar, Wireless internet access, Safe Deposit Box, Hair dryer, a balcony.
Designed for comfortable accommodation of 2 + 1 families, our room has a single bathroom. The total area of our rooms is 17 m2 with a total of 35 sea views.
There are 11 3-bed rooms on each floor, one on each corner and the usage area of these rooms is 20 m2. All rooms have sea view and a sofa for seating.
There are 1 specially designed honeymoon suites in our hotel. The hotel is a room with a view of the sea, which you can enjoy in the wedding ceremonies and the honeymoon holidays.
There are 2 rooms separated by an inner door. There are 2 single beds in the entrance and a large double bed in the second room.
There are 2 handicapped rooms in our hotel and 1 double bed in the room. The usage area of this room is 17 m2. Our room is designed according to disabled room standard of Ministry of Tourism.


text:
Menachem Elon (: מנחם אלון, žil 1. listopadu 1923 – 6. února 2013) byl izraelský právník a soudce, který v letech 1977 až 1993 působil jako soudce Nejvyššího soudu a zároveň v letech 1988 až 1993 jako místopředseda Nejvyššího soudu. V roce 1983 v prezidentské volbě neúspěšně kandidoval proti Chajimu Herzogovi.

Biografie
Narodil se v německém Düsseldorfu a v důsledku vzestupu nacismu v roce 1934 společně s rodinou emigroval do Nizozemska a o rok později do britské mandátní Palestiny. Studoval halachu (židovské náboženské právo) v Hebronské ješivě a vystudoval právo na Hebrejské univerzitě v Jeruzalémě, kde se později stal profesorem. Mimo jiné též působil jako hostující profesor na právnických školách Harvard University a New York University.

Napsal řadu děl na téma židovského náboženského práva a v průběhu svého působení v pozici soudce izraelského Nejvyššího soudu ve svých názorech často čerpal z principů židovského práva. Jeho soudní rozhodnutí zahrnují zákaz uvádění povahy neortodoxních konverzí do izraelských občanských průkazů a návrat dívky, která byla předána k adopci bez souhlasu rodičů.

Sehrál klíčovou roli ve hnutí Mišpat ivri (Hebrejské právo). Mezi jeho díla se řadí čtyřsvazkové dílo Jewish Law : History, Sources, Principles, zabývající se židovským právem, které je využíváno k akademickým účelům a pro výuku izraelských studentů práva. V roce 1979 mu byla za jeho počiny v oblasti práva udělena Izraelská cena.

V roce 1983 kandidoval s podporou pravicové strany Likud v prezidentské volbě proti Chajimu Herzogovi. Volbu prohrál poměrem hlasů 57 ku 61. Měl pět dětí, mezi něž patří bývalý člen Knesetu a ministr Binjamin Elon a někdejší ředitel Ješivat ha-Kotel rabín Mordechaj Elon.

Zemřel 6. února 2013 ve věku 89 let.

Odkazy

Reference

Externí odkazy

  My Jewish Learning – Articles By Menachem Elon

Narození v roce 1923
Narození 1. listopadu
Úmrtí v roce 2013
Úmrtí 6. února
Izraelští právníci
Soudci Nejvyššího soudu Státu Izrael
Vyučující na Hebrejské univerzitě v Jeruzalémě
Němečtí Židé
Absolventi Hebrejské univerzity v Jeruzalémě
Muži
Nositelé Izraelské ceny
Narození v Düsseldorfu

redpajama_set_name: RedPajamaWikipedia


text:
<#############################
| DETECTING CHARACTER CODE 0 |
##############################

Occasionally, strings use a "Byte 0" character as a delimiter. Unlike most other delimiters, this
delimiter does not show in text output but can still be used to separate the text parts.

PowerShell can deal with character code 0 strings. It is represented by a backtick followed by
the number 0. Note that text needs to be placed in double-quotes in order to convert the backtick
sequence to byte 0.

Here is an example illustrating how you split text that uses code 0 as delimiter.

#>

# create a sample text
$text = "Part1`0Part 2`0Part 3"

# delimiter does not show in output
$text

# ... but can be used t split:
$text -split "`0"

redpajama_set_name: RedPajamaGithub


text:
Covid-19 reaches Taloyoak; recoveries outpacing new cases across territory
by Derek Neary January 11, 2022
The number of active Covid-19 infections across Nunavut fell to 192 as of Tuesday, according to the Department of Health. Pixabay image
Taloyoak has a presumptive case of Covid-19 as of Tuesday, according to the Department of Health.
It's one of five new cases across the territory. However, there were 26 recoveries announced Tuesday, dropping the overall number of active Covid-19 infections to 192, from a high of 297 late last week.
Iqaluit remains the community with the most cases, at 55. There are 35 in Rankin Inlet; 22 in Kinngait; 21 in Arviat; 16 in Cambridge Bay; eight in each Pangnirtung, Sanirajak and Coral Harbour; seven in Whale Cove; four in each Qikiqtarjuaq and Chesterfield Inlet; two in Iglulik; and one in each Pond Inlet and Sanikiluaq.
The number of recoveries from the virus since the inception of this latest outbreak has climbed to 165.
"As predicted, Covid-19 is making its way into more of our communities, and affecting more and more Nunavummiut," said Dr. Michael Patterson, Nunavut's chief public health officer. "As we learn to live with the virus, we need to remember we know more and have more tools to stay well. Please continue to follow all the orders, get vaccinated and boosted when it's time, look out for one another, and remember that your actions have an impact on everyone's health."
Nunavummiut returning from holiday travel are reminded to self-isolate for 14 days if they are not triple vaccinated, to help contain the spread of Covid-19.
Anyone who develops Covid-19 symptoms – runny nose, cough, sore throat, fever or difficulty breathing – should call the Covid hotline at 1-888-975-8601 between 10 a.m. and 6 p.m. ET. There are longer wait times to get through to the hotline, so please remain patient. Please do not call or go to the health centre.
Derek Neary
Derek Neary has been reporting on developments in the North for 18 years. When he's not writing for Nunavut News, he's working on Northern News Services' special publications such as Opportunities North,...	More by Derek Neary

redpajama_set_name: RedPajamaCommonCrawl


text:
Stănești este un sat în comuna Poiana Vadului din județul Alba, Transilvania, România.

Stănești, Poiana Vadului


Everything was well prepared, especially the city tours with high technology support was super cool. We feel lucky that we chose Smallworld experience for our team building in Macau. Was beyond our expectation! It was such an amazing way to discover macau city center and historical site in a half day as well as enhance team cooperation. In all, I would recommend this to whoever I know that would like to have a team activity in Macau. Looking forward to new experience with Smallworld Experience again!


I missed carpool because I worked a little late, so I had to ride the BART, then take a city link bus to get to Vallejo. I got home around 8:45pm and was so tired. Once I walked in the house, I saw a package from Rosy Dawn Gardens. YEAY!!! … My Coleus…. I took photos, but they are still in my camera. I'll post them later today.
Before I post them, I wanted to show photos of the shelves I added in my bedroom to grow seedlings and overwinter cuttings. This photo was taken about a month ago.
Below is a new photo of the bottom shelf that mostly houses my Coleus. Some are grown from seeds that I collected last year and some are overwintered cuttings. Click on the photo to see some notes I added on with flickr.
This entry was posted in Uncategorized and tagged coleus, cuttings, growlights, seedling. Bookmark the permalink.


text:
Spotlight on Faculty
CCNY Social Media
City Beat Newsletter
From City to the World Podcast
CCNY study: advanced plastics recycling yields climate benefits
< Home / News / CCNY study: advanced plastics recycling yields climate benefits
The City College of New York Grove School of Engineering today released a new report which examined advanced recycling. The report concluded that advanced recycling helps avoid climate impacts, reduces demand for energy resources, and offers key tools for expanding the circular economy. The report was authored by Dr. Marco J. Castaldi, professor of chemical engineering and director of CCNY's Earth Engineering Center (EEC), and EEC research associate Lauren Creadore.
The authors examined 13 recently completed life cycle assessments (LCAs) and found that advanced recycling can transform hard-to-recycle plastics into products with a smaller carbon footprint than those made from new resources. The processes also reduce energy use and greenhouse gas emissions compared to conventional end of life methods, such as landfilling and waste-to-energy. Use of advanced recycling contributed to circularity for plastics in all 13 LCAs.
Specifically, the report found that advanced recycling technologies can:
Produce plastic and chemical products with a reduced global warming potential compared to products made from virgin resources.
Reduce the need for fossil energy resources by up to 97% compared to landfilling.
Reduce CO2 equivalent emissions by more than 100% compared to typical end-of-life processes when accounting for displaced demand for chemical products and energy.
"As advanced recycling becomes increasingly efficient, it is poised to play a major role in achieving global sustainability goals," said Castaldi. "It can transform hard-to-recycle plastics into a multitude of high-value feedstocks, reducing the need for fossil resources and limiting the environmental impact of waste management. Equally important, the data suggest that our transition to a more circular economy will dramatically improve climate outcomes."
"There's been significant investment and interest from companies and policymakers in advanced recycling," said Bob Hall at the American Society of Mechanical Engineers' Research Committee on Energy, Environment, and Waste. "Scientific studies like this one from CCNY, coupled with engineering ingenuity can help inform and responsibly move us towards a more sustainable and circular future. As these technologies mature, it is imperative to conduct additional studies on advanced recycling that adhere to international LCA standards."
"ASME suggests that technologies that have current operating performance data and have a facility to visit should be given priority for future analyses," added Annette Scotto, ASME Chair of the Material and Energy Recovery Division.
The report is titled "Quantitative Comparison of LCAs on the Current State of Advanced Recycling Technologies." Click here to read it in its entirety.
About the City College of New York
Since 1847, The City College of New York has provided a high-quality and affordable education to generations of New Yorkers in a wide variety of disciplines. CCNY embraces its position at the forefront of social change. It is ranked #1 by the Harvard-based Opportunity Insights out of 369 selective public colleges in the United States on the overall mobility index. This measure reflects both access and outcomes, representing the likelihood that a student at CCNY can move up two or more income quintiles. Education research organization DegreeChoices ranks CCNY #3 nationally for social mobility. In addition, the Center for World University Rankings places CCNY in the top 1.8% of universities worldwide in terms of academic excellence. Labor analytics firm Emsi puts at $1.9 billion CCNY's annual economic impact on the regional economy (5 boroughs and 5 adjacent counties) and quantifies the "for dollar" return on investment to students, taxpayers and society. At City College, more than 15,000 students pursue undergraduate and graduate degrees in eight schools and divisions, driven by significant funded research, creativity and scholarship. This year, CCNY launched its most expansive fundraising campaign, ever. The campaign, titled "Doing Remarkable Things Together" seeks to bring the College's Foundation to more than $1 billion in total assets in support of the College mission. CCNY is as diverse, dynamic and visionary as New York City itself. View CCNY Media Kit.
Jay Mwamba
e: jmwamba@ccny.cuny.edu


text:
After a post-holiday lull in new steel track being added to Wicked Cyclone's structure, the pace of work has picked up once again. And that means that we get to see the new extreme elements that are being added to the coaster's layout.
Above is a photo that the park shared, one of several, that shows off Wicked Cyclone's far side - near the New England SkyScreamer. As the trains head toward this section they will head through the 200 degree stall (middle of the photo) and into another heavily overbanked turn (left).
This view is also great as it really shows off how much Wicked Cyclone is shorter (in most areas, anyway) than before. But that's just fine, it means the elements will be encountered with even more speed, which leads to more forces and more fun!
The photos that Six Flags New England shared are good, but if you want some really great ones (great views of that 200 degree stall) click over to SFNEOnline's latest update.


Twitch has a lot of terrific events lined-up for creatives, viewers, cosplayers and gaming fans this year. Earlier today the company announced several major initiatives at TwitchCon 2016 for its Twitch Creative category, including a cosplay contest with a milestone-setting prize pool totaling $63,000. Other Creative happenings include art galleries, panels, and meet & greets with top broadcasters. Tickets for TwitchCon 2016 are currently available at Twitchcon.com.
Beginning today, broadcasters are invited to enter the cosplay contest by streaming their costume construction progress on Twitch Creative for a chance to win part of a $63,000 prize pool. The contest runs through August 22, 2016, with the top 20 finalists announced on September 9, 2016, and winning $1,500 each to travel to TwitchCon 2016. Four of the finalists will be selected from a community vote that will be active from August 26-29, while the category and overall winners will be determined at the conference. Attending the show and taking part in select phases of the judging process are popular cosplayers Yaya Han and Commander Holly, and streaming personality GiantWaffle, among others to be announced. The event will be emceed at TwitchCon 2016 by Anna Prosser Robinson.
In order to enter, participants must broadcast, archive and tag (#CosplayContest) a minimum of 15 hours of content via Twitch Creative by August 22, 2016. They will be competing in one of four categories–Needlework, Armor, FX, and Larger Than Life–with an overall winner also being selected. Finalists will get to show off their creations on the Kappa Stage at TwitchCon 2016 on Saturday, October 1 where the winners will be announced. The contest is only open to legal citizens of the United States and Canada (excluding Quebec) with full contest rules here.
Winners will receive medals and trophies hand-crafted by cosplayer and Twitch Creative broadcaster, Harrison Krix of Volpin Props. Watch the making of each award on Harrison's Twitch channel every Wednesday at 11:00am PT starting on June 29: twitch.tv/volpinprops. To see all of the TwitchCon 2016 cosplay entries in progress, visit the #CosplayContest community on Twitch Creative for a menu of all live broadcasts.
Twitch Creative Origin Story with Bill Moorier (Head of Twitch Creative), Kyle "Mos" (Creative community manager) and Darren "GeersArt" Geers (2D illustrator, Twitch): This panel charts the history of Creative on Twitch from its Justin.tv days to where the category is heading.
Twitch Creative has attracted so many talented artists and there will be a large gallery displaying a collection of the community's masterpieces with arts and crafts available for purchase in the Artist Alley. There will also be a number of other Creative broadcasters taking part in events, such as Dethskulpt, MissCoookiez and PianoImproman.
TwitchCon 2016 will take place September 30-October 2 at the San Diego Convention Center in San Diego, CA.


The world's top teams renew rivalries this summer in Russia. Featuring a thermally bonded seamless construction for a perfect flight, this football is the official match ball of the 2018 FIFA World Cup™. Inspired by Russia's urban landscapes, a pixelated graphic pays tribute to the iconic Telstar ball.


floating shelf for kitchen intended for Household Interior | Blueicejets.com floating shelves for a kitchen. floating shelves for kitchen walls. floating shelves next to kitchen cabinets.
Ana White Bigger Stronger Kitchen Floating Shelves Diy Projects Within Floating Shelf For Kitchen Decorating. How To Install Floating Kitchen Shelves Over A Tile Backsplash Intended For Floating Shelf For Kitchen Renovation. Floating Shelves For Kitchen Most Inspiring Floating Shelves Kitchen Inside Floating Shelf For Kitchen Plan. Floating Shelves For Kitchen Floating Shelves Kitchen Wood Kiwest Intended For Floating Shelf For Kitchen Renovation. How To Install Heavy Duty Floating Shelves For The Kitchen Pertaining To Floating Shelf For Kitchen Plan. Diy Heavy Duty Bracket Free Floating Kitchen Shelves House Updated Regarding Floating Shelf For Kitchen Prepare. Diy Heavy Duty Bracket Free Floating Kitchen Shelves House Updated Intended For Floating Shelf For Kitchen Renovation. Kitchen Floating Shelves Colors And Craft Intended For Floating Shelf For Kitchen Ideas. Floating Shelves For Kitchen Rustic Modern Kitchen With Floating Inside Floating Shelf For Kitchen Prepare. Chunky Diy Floating Kitchen Shelves With Regard To Floating Shelf For Kitchen Decorating. 20 Contemporary Floating Shelves In The Kitchen Home Design Lover Inside Floating Shelf For Kitchen Prepare. Diy Heavy Duty Bracket Free Floating Kitchen Shelves House Updated Throughout Floating Shelf For Kitchen Prepare. Kitchen Chronicles Diy Floating Rustic Shelves Jenna Sue Design Blog With Floating Shelf For Kitchen Renovation. Extra Long 10 Deep Rustic Industrial Floating Shelves Iron Pipe In Floating Shelf For Kitchen Prepare. How To Install Heavy Duty Floating Shelves For The Kitchen Intended For Floating Shelf For Kitchen Plan. 20 Diy Floating Shelves In 2018 Ghostly Decor Pinterest With Regard To Floating Shelf For Kitchen Plan. Gray Kitchen Cabinets With Floating Shelves Diamond With Regard To Floating Shelf For Kitchen Plan. 11 Kitchen Storage Spots You Completely Forgot About Cleaning Regarding Floating Shelf For Kitchen Plan.


Get a FREE Swiss Miss 3in1 Hot Cocoa Mix when you shop RCBC Bankard for at least P1,500 atLandmark or Sta. Lucia Supermarkets. Valid until August 31, 2015.
1. Promo period is from May 25 to August 31, 2015. Redemption is valid up to October 31, 2015.
s are active and current and in good credit standing.
3. To qualify, a cardholder must make a single-receipt purchase of AT LEAST Php 1,500 using his/her RCBC Bankard within the promo period and at any of the following participating supermarkets: Landmark Supermarket (Trinoma and Makati), Sta Lucia East Supermarket (Sta. Lucia East Grand Mall Phase 1 and 3).
4. For each qualified charge slip, RCBC Bankard cardholders can redeem for FREE three (3) sachet packs of Swiss Miss 3-in-1 Hot Cocoa Mix of these variants: Milk Chocolate, Dark Chocolate, Marshmallow.
5. Cardholders may redeem free Swiss Miss from the RCBC Bankard sales booths or Customer Service counters located at the participating supermarket outlet where valid purchase was made and during regular store hours.
6. To redeem, a cardholder must present his/her RCBC Bankard credit card and original credit card charge slip (Customer's copy) for proper validation at participating supermarket outlet. Cardholder information and transaction details on the charge slip must comply with the promo mechanics and must match with the RCBC Bankard credit card presented.
7. Redemption must be done at the partner supermarket where purchase was made.
8. Redeemed charge slip/s must be original and will be surrendered to participating supermarket outlet and may not be used anymore for other promotional offers. Cardholders may opt to photocopy their charge slip/s prior redemption, for their personal file.
9. Splitting of a single transaction for the purpose of generating multiple charge slips is not allowed. Redemptions from such charge slips will be deemed fraudulent.
10. Authorized representatives are not allowed to redeem.
11. RCBC Bankard reserves the right to charge the cost of the redeemable product to a cardholder's RCBC Bankard should the latter cancel the qualifying transaction, or if the cardholder's redemption is found to be fraudulent.
12. RCBC Bankard is not obliged to recognize or replace any unissued, misplaced, lost, stolen or faded charge slips.
13. Redeemable products under the promo may not be converted to cash, credited to RCBC Bankard or to any other product/s or promo offers available at participating supermarket outlet.
14. Any dispute concerning the free products shall be settled directly between the cardholder and the manufacturer, ConAgra Foods International, with assistance from RCBC Bankard.


Musser's All In One Tire & Auto Center provides Engine Tune-Up services to Tipp City, OH, Troy, OH, Vandalia, OH, and other surrounding areas.
Why Should You Have Engine Tune-Up Services Performed at Musser's All In One Tire & Auto Center?
We proudly service the Engine Tune-Up needs of customers in Tipp City, OH, Troy, OH, Vandalia, OH, and surrounding areas.


text:
Highland Sky
Bring Me Home
CountryAmericana
For fans of:Dixie Chicks, Sheryl Crow, Michelle Branch, Sarah Buxton, Taylor Swift
Bring Me Home.mp3
Change.mp3
Cry.mp3
Let Go.mp3
My Turn.mp3
From the buzzing lights of Hot Springs, Arkansas to the stages of Nashville, comes the sister sensation known as Highland Sky. This duo compiles elements of early Dixie Chicks, Michelle Branch, and the soulful honesty of Sheryl Crow. With their "Bring Me Home" EP now on iTunes and shows on the calendar, Highland Sky is a homegrown experience that has real, raw talent. The sisters grew up in Hot Springs National Park, Arkansas where they began performing from an early age on stage together at their family's music and variety show "The Bath House Show" (1988-2008). Featuring music from the 1930s through today, the show was a huge success by bringing family fun and entertainment to more than 2,000,000 patrons and locals combined in its time. You can currently find the duo performing at different venues in Nashville, TN.
Counting Crows 2012-08-24T00:00:00-07:00
The Mountain Goats 2011-10-27T00:00:00-07:00
Hot 8 Brass Band 2017-04-18T00:00:00-07:00


The woman behind the warm-up routine at Stamford's Race for Life has revealed how losing her dad to cancer has made her determined to make this year's event the best yet.
Sonia Cullington, 44, has helped ladies limber up for the 5k and 10k Stamford races for years, but this time the event will be extra special as she will lead the warm-up in memory of her father Ken Nicoll, who died of cancer last month.
The mum-of-one, from Oundle, is looking forward to seeing other women join her in the fight against the disease in the event, which takes place at Burghley House on Sunday.
Her father's cancer was diagnosed by chance - he did not feel ill but sent back a sample test that came through the post. He was called in for further analysis and bowel cancer was diagnosed. He underwent three operations and chemotherapy but sadly the cancer spread.
Sonia said: "He was fighting it and fighting it until the end. He wanted to live, he wanted to prolong his life but in December, just before Christmas, dad's GP called and asked my sister and me to come to the house. That was when he started to use the word terminal.
"It was a shock, I hadn't heard him use that word – terminal. I had to think, where do we go from here and how can I help? It was very difficult. To watch a family member go through it is agonising. I just had to keep being positive and see what I could do to help.
"It will be emotional when I am on the stage getting people warmed up but it means a lot to me to do it.
Cancer Research UK events manager Julie Braithwaite said: "Sonia has been an amazing supporter of our Stamford Race for Life event and has always made sure our ladies are ready to take part.
The event starts at 11am and supporters are welcome to cheer on the ladies at Burghley Park.
l To find out more, turn to page 41.


text:
What was the cutting built for?
The Miramar wharf was built in 1901, promoted by C.J Crawford and A.D, with other landowners of course, were anxious to give Wellington buyers at their land sales.
The Miramar ferry company was formed and ran the two ferries, Royalty and Admiral between the city and three wharves on the Miramar Peninsula. From the Wharf, a deep cut was dug to make easy access to the land ahead. It was also built to make repairing boats easier, with the dock being so close.
By Luka, Year 6
The cutting at Miramar. Smith, Sydney Charles, 1888-1972 :Photographs of New Zealand. Ref: 1/1-020294-G. Alexander Turnbull Library, Wellington, New Zealand. http://beta.natlib.govt.nz/records/23118326


text:
RWE Group EBITDA decreased 2% to €7 billion
Staff writer ▼ | March 9, 2016
The RWE Group was unable to match the operating result from the previous year, but nonetheless achieved its earnings targets despite unfavourable framework conditions in the energy sector.
RWE Group The successful disposal of RWE Dea
The 2% decline in EBITDA compared to the previous year, to €7 billion, was actually smaller than expected. This can be attributed to special items such as the full consolidation of the Slovak energy utility VSE from the end of August.
Another reason for exceeding the target was one-off income realised for insurance claims for the power plant project at Hamm. Both of these items had a positive effect on EBITDA and the operating result, although the decision not to complete the construction of Block D required the company to recognise an impairment of €654 million, which was included in the operating result.
At €3.8 billion (4% below the previous year), the operating result was in line with expectations, as was adjusted net income which amounted to €1.1 billion (12% down on the previous year).
In fiscal 2015, the RWE Group passed a number of important milestones in terms of boosting its financial strength. Net debt was down almost one-fifth, to €25.1 billion. The main reason for this was the successful disposal of RWE Dea, which had an impact of €5.3 billion, including the interest on the sale price.
Further disposals had a total debt-reducing effect of €1.4 billion. The increase in the discount rates used to calculate provisions for pensions, which reflect the recent development of market interest rates, also helped reduce debt.
Good progress was also made in implementing the current efficiency-enhancement programme: a lasting effect on the operating result of €1.6 billion was achieved by the end of 2015, which was €100 million more than the original target.
The company is therefore planning new steps to increase its operating effectiveness, focusing on conventional power generation and the UK supply business, which it intends to comprehensively restructure. In total, the Group aims to achieve a positive impact of €2.5 billion on the operating result (previously €2.0 billion), which should be fully felt from 2018 (previously from 2017).
The strategic restructuring of the RWE Group, which was decided upon at the end of 2015, is also on schedule.
The new subsidiary bundling renewables, grids and retail both in Germany and internationally, will begin operating on 1 April 2016.
Looking back at 2015, CEO of RWE AG Peter Terium noted, "Last year we had to deal with a number of difficulties, ranging from discussions on energy policy to problems in our UK supply business and the further decline in prices for wholesale power trading.
"But I am proud of the efforts put in by our employees, since we have collectively kept the RWE Group on track. This has meant making a number of tough decisions. As a result, we have opened up new business opportunities. However, energy policy challenges still continue to pose a burden." ■
TeliaSonera Q2 net sales decreased 1.2%
TeliaSonera net sales increased 6.3 percent
Telenor Q1 revenues decreased 3%
11.5 million of Orange shares will be offered to employees
Automatic exchange of tax-related financial data in EU
Navistar Q4 net income $102 million
Bed Bath and Beyond Q3 net loss 38.6 million
CHS reports $177.9 million in Q1 net income