Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria.
To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host.
The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.
Plant biomass recalcitrance is one of the most important barriers to its use as a substrate for the production of fuels and chemicals using microorganisms as catalysts. The plant cell wall consists of a complex web of polysaccharides and phenolics that function in plant structure and development . Perennial plants like switchgrass could be incorporated into so-called agro-ecosystems, which would increase carbon storage and biofuel production, decrease carbon dioxide emissions, and improve water quality through wetland denitrification . While the natural recalcitrance of plant biomass is a major barrier , several methods including direct microbial breakdown of cell wall structures can be used to liberate energy-rich sugars for conversion to useful biofuels and bioproducts.
Chemical and thermal pretreatments are often used to break down the raw substrate, but they are expensive and destructive to the sugars in the biomass , and they produce hydrolysates inhibitory to both cellulose degradation and microbial growth . In contrast, thermophilic anaerobes in the genus Caldicellulosiruptor can deconstruct high loadings of plant biomass into simple sugars without conventional pretreatment - and have recently been engineered to produce ethanol directly from switchgrass . Caldicellulosiruptor species can simultaneously utilize the wide range of hexoses, pentoses, oligosaccharides, and polysaccharides released from the plant cell wall, and there is no evidence of carbon catabolite repression ,. These qualities make them well suited for consolidated bioprocessing (CBP), in which one microorganism is used for both biomass deconstruction and end-product formation.
Members of the Caldicellulosiruptor genus are anaerobic Gram-positive bacteria, and they are the most thermophilic cellulose-degrading organisms known . They secrete free carbohydrate-active enzymes (CAZys)  with carbohydrate-binding modules that are well suited for binding and degrading cell wall polysaccharides . C. bescii is one of the strongest crystalline cellulose degraders in the genus, whereas the closely related C. hydrothermalis is one of the weakest . Interestingly, C. hydrothermalis lacks the multidomain CAZys found in more cellulolytic members of the genus  as well as a cluster of pectinases that affect C. bescii growth on biomass (D. Chung, submitted). In addition to lacking multidomain enzymes, C. hydrothermalis completely lacks the glycosyl hydrolase (GH) domains GH9 and GH48 , the domains that comprise CelA, the most highly secreted cellulase in C. bescii. C. hydrothermalis thus provides a "blank slate" with which to study thermophilic enzymes important for biomass degradation in vivo, and is a promising system for heterologous expression of plant biomass deconstruction enzymes.
To establish methods for genetic manipulation of C. hydrothermalis, we took an approach similar to the one previously used for C. bescii that relied on the selection of a pyrF deletion mutant, which allows for nutritional selection of transformants . Interestingly, C. hydrothermalis contains fewer mobile genetic elements than other members of the genus , so this species may have other advantages for genetic manipulation, including fewer issues with genome stability that could result from genetic selections and counter-selections. We transformed the pyrF deletion mutant with the pJGW07 shuttle vector that is based on a native plasmid, pBAS2, from C. bescii. Both the C. bescii and the heterologous Clostridium thermocellum wild-type pyrF allele were shown to complement this deletion, restoring the mutant to uracil prototrophy. Deletion of the ChyI restriction enzyme in C. hydrothermalis, a homolog of a HaeIII-like restriction enzyme known to be a barrier to transformation in C. bescii,, increased the transformation efficiency by about an order of magnitude. The new strain C. hydrothermalis JWCH008 should facilitate the assessment of plant biomass deconstruction by the Caldicellulosiruptor genus and the molecular engineering of deconstruction enzymes.
The pyrF gene encodes orotidine monophosphate decarboxylase, an enzyme in the pyrimidine biosynthesis pathway. Deletion of this gene results in uracil auxotrophy and resistance to 5-fluoroorotic acid (5-FOA), allowing prototrophic selection of transformants and counter-selection of the wild-type allele . The optimal growth temperature for C. hydrothermalis is 65°C , and we had previously observed an increase in the spontaneous mutation rate in cells grown above and below 65°C. To obtain spontaneous deletions of pyrF, cells were grown at non-optimal temperatures in the presence of uracil. The presence of uracil in the growth medium allowed for the maintenance of cells with spontaneous mutations in the pyrF gene. After growth at various temperatures, cells were plated onto a medium with 5-FOA selecting resistance and loss of pyrF function. One 5-FOA resistant mutant, JWCH006 (Table 1), that had been grown at 60°C contained a 99-bp deletion in pyrF (Figure 1A) and was confirmed to be a uracil auxotroph resistant to 5-FOA.
Isolation of a spontaneous pyrF mutant in C. hydrothermalis . (A) Chromosomal map of the uridine monophosphate (UMP) biosynthetic gene cluster in C. hydrothermalis JWCH006. The 99-bp spontaneous deletion in ΔpyrF is indicated by the line below the diagram. 462 bp lie upstream and 357 bp lie downstream of the deletion. Bent arrows depict primers used to verify the structure of the pyrF gene in the JWCH006 strain. (B) Gel depicting PCR products of the pyrF region in the wild-type strain (1.13 kb) compared to the JWCH006 strain (1.02 kb) amplified by the indicated primers (DC163 and DC164). M: 1 kb DNA ladder (NEB); 1: wild-type genomic DNA; 2: JWCH006 genomic DNA; 3: negative control.
Electrocompetent C. hydrothermalis JWCH006 (ΔpyrF) cells were prepared based on the method for C. bescii and transformed with a previously described shuttle vector, pDCW89 , containing the wild-type C. bescii pyrF allele. Transformation of C. bescii with plasmid DNA isolated from E. coli requires in vitro methylation by a methyltransferase, M.CbeI . As C. hydrothermalis has a similar restriction profile to that of C. bescii, we anticipated that in vitro methylation by M.CbeI would protect plasmid DNA isolated from E. coli and allow transformation of C. hydrothermalis. pDCW89 DNA methylated with M.CbeI in vitro successfully transformed the C. hydrothermalis ΔpyrF mutant to prototrophy at an average frequency of 37 colony forming units (CFUs) per microgram of DNA (Table 2).
The suffix M denotes plasmid methylated with C. bescii M.CbeI methyltransferase.
Results represent the average ± standard deviation of three biologically independent transformation experiments.
Since the deletion of pyrF in JWCH006 was only 99 bp (Figure 1A), and there is 95% DNA sequence homology between the pyrF genes in C. bescii and C. hydrothermalis, there was a large region of residual homology between the pyrF locus in the C. hydrothermalis chromosome and the C. bescii pyrF gene on the plasmid. Although pDCW89 DNA methylated in vitro with M.CbeI transformed C. hydrothermalis JWCH006 to prototrophy, we found that the wild-type plasmid-encoded pyrF allele repaired the mutant pyrF locus in almost every case (Additional file 1: Figure S1), and the plasmid was not maintained autonomously over time. Since marker replacement in C. hydrothermalis relies on the integration of non-replicating plasmids at sites other than pyrF, the C. bescii pyrF cassette in pDCW89 was replaced by the C. thermocellum homolog (Cthe0951) to create pJGW07 (Figure 2A). While the two PyrF protein sequences are 45% identical, the C. thermocellum pyrF gene has very low DNA sequence homology with the Caldicellulosiruptor pyrF genes. Transformation of JWCH006 with methylated pJGW07 was successful (Figure 2B, Additional file 1: figure S2A), and uracil prototrophic transformants were obtained at a frequency of 22 ± 10 CFU per microgram of DNA (Table 2).
Plasmid pJGW07 isolated directly from C. hydrothermalis Δ pyrF . (A) pJGW07 constructed by replacing the C. bescii pyrF gene with the C. thermocellum ATCC 4705 homolog Cthe0951. The hatched region was derived from C. bescii native plasmid pBAS2. AprR, apramycin resistance casette; repA, replication initiator for pSC101 replication origin; par, partitioning locus. (B) Agarose gel depicting pJGW07 plasmid DNA extracted from different sources. M, molecular weight standards; E, pJGW07 purified from E. coli; C, pJGW07 purified from C. hydrothermalis.
Plasmid pJGW07 was purified directly from C. hydrothermalis based on a previously published method for other Gram-positive organisms . Undigested plasmid isolated from C. hydrothermalis migrated in an agarose gel slightly differently from plasmid DNA isolated from E. coli, and we suggest that the difference is not in size but in methylation within the native host compared to E. coli (Figure 2B). Endonuclease restriction analysis using a panel of enzymes indicated that plasmid isolated from C. hydrothermalis is protected at GGCC sites by a HaeIII-like modification system as expected but not at GATC sites (Additional file 1: Figure S3). This suggests that C. hydrothermalis lacks the DNA adenine methylase present in E. coli. EcoRI or HhaI recognition sites are not protected in either organism.
To confirm that the plasmid was replicating autonomously, DNA isolated from the C. hydrothermalis transformant JWCH009 (Table 1) was back-transformed into E. coli DH5α. Plasmid DNA recovered from 12 apramycin-resistant E. coli transformants was identical in its restriction patterns to pJGW07 transformed into C. hydrothermalis, suggesting that the plasmid was structurally stable during transformation, replication, and back-transformation to E. coli (Additional file 1: Figure S2). There was no evidence of plasmid integration (Additional file 1: Figure S4) resulting from recombination between the C. thermocellum pyrF gene on the plasmid and the C. hydrothermalis pyrF gene in the chromosome.
These data show that, although C. thermocellum has an optimal growth temperature of 60°C, the C. thermocellum orotidine-5'-phosphate decarboxylase functions at temperatures up to at least 65°C and that the C. thermocellum gene is expressed at sufficient levels to complement the C. hydrothermalis pyrF deletion.
We recently reported the construction of a shuttle vector for C. bescii based on the smaller of two native plasmids in that species . The native plasmid pBAS2 is maintained in C. bescii at a copy number of approximately 75 . Because the shuttle vector derived from this plasmid competed with the native plasmid it was derived from, the shuttle vector was maintained in low copy and was readily lost without selection. While an unstable plasmid is useful for some applications, stability and high copy number also have advantages. No native plasmid DNA was detected in C. hydrothermalis using conventional plasmid isolation methods, nor was one identified during the sequencing of total DNA isolated from this strain . We anticipated that the C. bescii shuttle vector would likely replicate in C. hydrothermalis and might be stably maintained at a high copy number. Quantitative polymerase chain reaction (qPCR) was performed as described in the Methods section to determine the copy number of the pJGW07 plasmid in C. hydrothermalis, and the results indicated that it was maintained between 25 and 115 copies per chromosome over five serial transfers, but was quickly lost without selection (Figure 3, Additional file 1: Table S1).
Determination of plasmid copy number. Quantitative PCR (qPCR) was used to detect the copy number of plasmid pJGW07 in relation to the chromosome. Shown are the results after the fifth passage through A) selective and B) non-selective media. The x-axis is the number of iterations of the polymerase chain reaction, and the y-axis displays the logarithm of ΔRn, which is the fluorescence of the SYBR green dye with the baseline fluorescence subtracted. The number of cycles required to cross a given threshold (cycles to threshold or Ct) is reflective of the plasmid copy number (PCN). The threshold is indicated by a horizontal line. PCN was calculated using the formula PCN = 2^| Ctchromosome— Ctplasmid |. The copy number ranged from about 25 to 115 copies per chromosome in the cultures with selective media. The copy number was determined based on two independent biological samples with three technical replicates (Additional file 1: Table S1).
In developing methods for DNA transformation of C. bescii, we observed that restriction by a HaeIII isoschizomer, CbeI , was an absolute barrier to transformation of DNA from E. coli. We identified, cloned, expressed, and purified its cognate methyltransferase, M.CbeI, from C. bescii and showed that DNA methylated in vitro readily transformed C. bescii. Deletion of cbeI in C. bescii relieved the requirement for in vitro methylation of plasmid DNA from E. coli by M.CbeI  and allowed efficient DNA transformation. The C. hydrothermalis chyI gene is an ortholog of cbeI with 96% DNA sequence identity and 100% sequence coverage. A deletion of chyI was constructed on a plasmid, pJGW03, which was transformed into C. hydrothermalis JWCH006 (Figure 4A). A deletion mutant was readily obtained in a screen of 50 colonies, and only two rounds of purification on low osmolarity defined (LOD) plating media containing uracil  were required to resolve the merodiploid, resulting in C. hydrothermalis JWCH008 (ΔpyrF ΔchyI) (Figure 4C). We note that the region of the C. hydrothermalis genome that contains ChyI is not identical to the region of the C. bescii chromosome that contains CbeI. The C. hydrothermalis region contains an additional open reading frame (ORF) that apparently encodes a truncated form of the N-terminal portion of the ChyI protein. Sequence analysis revealed a premature stop codon likely resulting from a point mutation in the ChyI ORF. This altered gene structure had no obvious effect on enzyme function in vivo. The deletion we designed included both ORFs.
Deletion of the gene encoding putative restriction enzyme ChyI. A) Scheme for targeted gene deletion. The pJGW03 chyI knockout vector is transformed into JWCH006 (ΔpyrF), and uracil prototrophy selects for integration at one of the 500-bp flanking regions, denoted by the gray boxes. 5' integration is shown. The uracil prototroph is then plated on 5-FOA to select for loop-out of the pyrF cassette via homologous recombination between flanking region sequences. The two possible results are a return to the wild-type sequence or a clean chyI deletion. (B) Chromosomal map of the locus containing the ORF for chyI. The deleted region is indicated by the line below the diagram. Bent arrows depict primers used to verify deletion of chyI in the JWCH008 strain. (C) Gel depicting PCR products of the chyI region in the JWCH006 ΔpyrF strain (2.3 kb) compared to the JWCH008 ΔpyrF ΔchyI strain (1.25 kb) amplified by the indicated primers (JG025 and JG028). 1: C. hydrothermalis JWCH006 genomic DNA; 2: C. hydrothermalis JWCH008 genomic DNA; 3: negative control; M: 1 kb DNA ladder (NEB).
To assess whether the loss of the ChyI restriction enzyme resulted in an increase in transformation efficiency in C. hydrothermalis, we compared the transformation efficiencies of the two strains generated in this study, JWCH006 (ΔpyrF) and JWCH008 (ΔpyrF ΔchyI). As shown in Table 2, electrotransformation of the JWCH006 parent strain with either the pJGW07 shuttle vector containing the C. thermocellum pyrF gene, or the pDCW89 vector containing the C. bescii pyrF gene, was low but detectable, and methylation of plasmid DNA did not make a significant difference in transformability. The standard deviation in experiments with low numbers of transformants is substantial but not unexpected . This extremely low transformation efficiency may be an underestimate of the actual efficiency, as the plating efficiency of C. hydrothermalis on a selective solid medium is less than 10-4 (plating 106 cells as determined by cell count resulted in fewer than 100 colonies). Transformation of JWCH008 containing the chyI deletion was an order of magnitude higher than that of JWCH006, and again, there is no significant difference between the transformation efficiency of methylated and unmethylated DNA, suggesting differences in the restriction/modification systems of C. hydrothermalis and C. bescii. The fact that methylation with M.CbeI made no difference in transformation efficiency was somewhat surprising, especially considering the fact that deletion of ChyI increased the transformation frequency of unmethylated DNA. We interpret this to indicate that there are differences between C. bescii and C. hydrothermalis in their restriction/modification systems and perhaps additional enzymes in one or the other that account for the differences in transformation frequencies. It is also possible that the truncated form of the CbeI/ChyI orthologous proteins makes a difference in their activities (Figure 4B). We previously reported that C. hydrothermalis chromosomal DNA is resistant to digestion by BamHI and BspEI . Both these enzymes have six base recognition sequences that are relatively rare compared to four base recognition sequences. The plasmids used in this study do not contain BamHI sites. While there are two BspEI restriction sites (TCCGGA), E. coli DH5α, the strain we used to make plasmid DNA, contains an adenine methyltransferase known to protect this site and may prevent cleavage by C. hydrothermalis during DNA transformation.
We emphasize that the observed transformation of the JWCH006 parent strain is not an indication that C. hydrothermalis is naturally competent for DNA uptake. Preparation of electrocompetent cells and electroporation was necessary to detect transformation. We have invested some effort to induce natural competence in members of this genus, but those efforts have not been successful. There are homologs to the natural competence genes comEA, comEC, comGC, and dprA, but in contrast to other thermophilic anaerobes , there is no evidence to date of natural competence in Caldicellulosiruptor species.
Methods for genetic manipulation of C. hydrothermalis, based on those used for C. bescii, were successful and efficient. Restriction of DNA was not an absolute barrier to transformation, but deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude. Heterologous expression of the Clostridium thermocellum pyrF gene was sufficient to complement the C. hydrothermalis ΔpyrF mutant, allowing both autonomous plasmid replication at relatively high copy (about 25 to 115 copies/chromosome) and marker replacement of the chyI gene in the C. hydrothermalis chromosome. The use of this new strain, C. hydrothermalis JWCH008, should allow for the expression of heterologous and homologous enzymes for both the identification and analysis of enzymes involved in biomass deconstruction of unpretreated plant biomass by the Caldicellulosiruptor genus. It will also enable the engineering of glycosyl hydrolases such as CelA and other important plant biomass deconstruction enzymes in a strain devoid of similar enzymes or activities.
Wild-type C. hydrothermalis DSM 18901 was grown from a 0.5% inoculum in 50 mL of a low osmolarity defined growth medium (LOD) supplemented with 40 μM uracil. Cultures were grown at 55°C, 60°C, 68°C, and 75°C. Cells in the late exponential phase were cooled to room temperature, harvested by centrifugation at 6,100 × g, and resuspended in 1X C. bescii partial base salts . 100 μL of resuspended cells were mixed with a 1.5% agar overlay and plated onto LOD media with 40 μM uracil and 8 mM 5-FOA. The plates were degassed in anaerobic chambers and incubated for 4 days at 68°C. The colonies were picked directly into 20 mL LOD media with 40 μM uracil, which was immediately degassed and incubated at 68°C. When the media was turbid, chromosomal DNA was extracted with a Zymo Research gDNA Extraction kit (Irvine, CA). The pyrF gene region was amplified from the wild type and deletion mutant with primers DC163 and DC164 (Figure 1A), and the PCR products were analyzed for the presence of deletions in a 1.5% w/v agarose gel by electrophoresis (Figure 1B). All plasmids were verified by DNA sequencing (GENEWIZ).
High-Fidelity Q5 Polymerase (New England Biolabs (NEB), Ipswich, MA), restriction enzymes (NEB), and Fast-Link™ DNA Ligase (Epicentre Technologies, Madison, WI) were used according to manufacturer instructions. pJGW07 (Figure 2A) was constructed by replacing the C. bescii pyrF gene (Cbes1377) on the replicating shuttle vector pDCW89  with the Clostridium thermocellum homolog for pyrF (Cthe0951). Cthe0951 was amplified by PCR using primers JG021 and JG022, engineered to contain XbaI and NcoI restriction sites, respectively. This 945-bp PCR product was ligated directionally to a 6.75-kb pDCW89 PCR product amplified using primers JG023 and JG024, which also contain XbaI and NcoI sites. Correct clones were purified from E. coli with a Miniprep kit (Qiagen), and confirmed by restriction fragment analysis and DNA sequencing (GENEWIZ).
To prepare cells for transformation, 15 mL of a freshly grown JWCH006 (ΔpyrF) culture were inoculated into four 500-mL bottles of fresh LOD supplemented with 40 μM uracil and 19 amino acids, and incubated at 65°C to the early exponential phase (OD680 approximately 0.04 to 0.05). The cultures were cooled to room temperature for 1 h, harvested by centrifugation (5,000 × g, 10 min) at 4°C and washed three times with 50 mL pre-chilled 10% sucrose. After the final wash, the cell pellets were resuspended in about 250 μL pre-chilled 10% sucrose. 60-μL aliquots of competent JWCH006 were added to plasmid DNA (0.5 μg), either methylated with M.CbeI methyltransferase, as previously described , or unmethylated, gently mixed, and incubated in 10% sucrose for 15 min at room temperature. Electrotransformation of the cell/DNA mixture was performed via a single exponentially decaying electric pulse (1.8 kV, 350 Ω, and 25 microF) in a pre-chilled 1-mm cuvette using a Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA). After pulsing, the cells were incubated in 10 mL low osmolarity complex growth medium (LOC)  at 65°C. After 4 h, the cultures growing in LOC medium were cooled to room temperature, harvested by centrifugation (6,100 × g, 10 min), and washed three times at room temperature with 1X C. bescii partial base salts  to remove the rich media. The cells were finally resuspended in 800 μL 1X base salts. For each plate, 100 μL of resuspended cells were mixed with 2 mL of a 1% agar overlay and plated onto LOD media lacking uracil to select for transformation. The plates were degassed in anaerobic chambers and incubated for 4 days at 65°C. The colonies were picked directly into 20 mL LOD media without uracil, which was immediately degassed and incubated at 65°C. Uracil-prototrophic transformants were confirmed by PCR amplification of C. hydrothermalis DNA using primers JG021 and JF199, which are specific for pJGW07 (Figure 2C, Additional file 1: figure S2A).
A single transformant colony was picked directly into 20 mL LOD media lacking uracil, which was immediately degassed and incubated at 65°C. This strain maintaining the pJGW07 shuttle vector was named JWCH009. JWCH009 was grown to the late exponential phase (OD680 approximately 0.15) in 50 mL LOD. Direct extraction of plasmid DNA from JWCH009 was performed as previously described ,. The plasmid DNA was digested with enzymes HaeIII, EcoRI, HhaI, and MboI (NEB).
To determine the structural stability of the plasmid, DNA was extracted from C. hydrothermalis with a gDNA Extraction kit (Zymo Research), and 2 μL of DNA was electrotransformed into E. coli DH5α via single electric pulse (2.5 kV, 200 Ω, and 25 microF) in a pre-chilled 2-mm cuvette using a Bio-Rad Gene Pulser. The cells were placed into 1 mL SOC media for 1 h with shaking at 37°C, and then plated onto LB agar supplemented with 50 μg/mL apramycin. The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen) and screened with restriction enzymes EcoRI and ApaLI (NEB) (Additional file 1: Figure S2B).
To determine the pJGW07 copy number and maintenance over time, JWCH009 was grown to OD680 approximately 0.15 and serially subcultured for 5 days. The total DNA was isolated from the cultures and treated with RNase A. qPCR experiments were carried out with a LightCycler 480 Real-Time PCR instrument (Roche, Indianapolis, IN) with LightCycler 480 SYBR Green I Master mix (Roche). Two independent sets of primers specific to either the pJGW07 plasmid (Q1/Q2 inside Cbes2777, Q3/Q4 inside Cbes2778) or the C. hydrothermalis chromosome (Q11/Q12 inside Calhy0897, Q13/Q14 inside Calhy1377) were used to compute the relative copy number of the plasmid to the chromosome. Three replicate reactions for each primer set were performed, and the average of the two primer sets on each sample was used to calculate the plasmid copy number in each serial subculture (Additional file 1: Table S1) according to the method of Lee et al.. The amplification efficiency over a 104-fold range of DNA concentrations was 93.5%, within the ratio of 90 to 110% considered acceptable (Life Technologies).
A vector backbone was amplified from suicide vector pDCW88  with primers DC081 and DC262. Flanking regions for the chyI gene (Calhy0408) were amplified using primers DC484 and DC485 (5' flanking region) and DC486 and DC487 (3' flanking region). The 5' and 3' flanking regions were combined into one fragment by overlap extension PCR and ligated into the pDCW88 vector backbone using restriction enzymes KpnI and ApaLI. The resulting plasmid pDCW151 was used to construct pJGW03 in which the C. bescii pyrF gene cassette (Cbes1377) was replaced with the C. thermocellum pyrF gene (Cthe0951) as described above. Competent C. hydrothermalis JWCH006 cells were prepared as described above. 1.0 μg of non-replicating plasmid pJGW03 was added to 50-μL aliquots of competent JWCH006, gently mixed, and incubated for 15 min at room temperature. Electrotransformation of the cell/DNA mixture was performed via a single electric pulse (1.8 kV, 350 Ω, and 25 microF) in a pre-chilled 1-mm cuvette using a Bio-Rad Gene Pulser. After pulsing, the cells were incubated in 10 mL low osmolarity complex growth medium (LOC)  at 68°C with shaking at 150 rpm. From this culture, 0.5% inocula were transferred into LOD medium lacking uracil at 65°C every hour for 4 h to select for integration into the C. hydrothermalis genome. When the cultures in the selective media were turbid, a 0.5% inoculum was transferred to 50 mL LOD supplemented with 40 μM uracil to allow a loopout of the wild-type pyrF allele to occur. This culture was then plated onto LOD plates supplemented with 40 μM uracil and 6 mM 5-FOA to select against the pyrF wild-type allele, and grown for 4 days at 68°C. Colonies were picked directly into 20 mL LOD medium with uracil, which was immediately degassed and incubated at 68°C. The resulting cultures were screened for a deletion using primers JG025 and JG026, and one deletion culture was purified twice on solid LOD media supplemented with 40 μM uracil.
JG carried out experiments, led the work and contributed to the writing of the manuscript. DC carried out experiments, contributed to the design of experiments and contributed to the writing of the manuscript. JY carried out experiments and contributed to the writing of the manuscript. JW participated in the design of the experiments, led the work and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
We thank Jennifer Copeland for her expert technical assistance. JG was supported by a Presidential Fellowship from the University of Georgia Research Foundation. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.


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Chevrolet Matiz
For several years, the Chevrolet Matiz is a cheap alternative in the small car class. The 1.5 meter wide car is ideal as a city car. As with the predecessor produced until 2005 (which was also known as the Daewoo Matiz, you can select the current Chevrolet Matiz two different engine variants : In the 1.0-SX version of the Chevrolet small car reaches a top speed of 156 km / h with an acceleration of 14.1 seconds from 0 to 100 km / h For the Chevrolet Matiz 0.8 with 52 HP, however, 145 km / h top and 18.2 seconds from 0 to 100 km / h can be specified. Daf ÜR is the Korean built mini city car with an average consumption of 5.6 liters very frugal. With its large circular lights, which still emphasize the tailgate, the five-door lifts visually from the predecessor. Inside the car, the Matiz especially the unusual positioning of the instrument stands out: These are now done on the center console and not, as usual, in front of the driver. The standard features of the Chevrolet Matiz SX is very neat. Among other things, are front and side airbags, power steering, fog lights, central locking, roof rails, air conditioning, CD radio and electric windows contain. An optional extra the agile city car will be offered with an automatic transmission.
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redpajama_set_name: RedPajamaCommonCrawl


We strongly encourage our pupils to pursue a range of interests at school and to develop an understanding of the wider world both local and global. Dore is keen to extend the education we offer beyond the classroom, seeking wider partnerships to extend our knowledge, understanding and global citizenship responsibilities. We offer numerous extra activities at lunch times, after school, educational visits and visitors and residential trips for pupils to participate in. Read about these and our annual focus events such as Send My Friend to School, Children in Need, Anti-bullying week and many more. See below the News button for Special events and the News archives.
The Junior Concert this year featured Y6 actors and a large mixed choir starring in a musical version of A Midsummer's Night Dream. Many thanks go to all the cast and choir for their hard work in preparing for this wonderful concert during lunch-times and after school. This brilliant project was led by, Mrs. Irene Warburton, Miss Heather Atkinson and Mr. Ian Faraday and we are very grateful to Mrs. Helen Leach who led on the costumes and Mrs. Claire Tinker who supported with scenery.
The show was absolutely fantastic and it was brilliant to see Shakespeare performed in this way by such a youthful cast. It looked and sounded engaging and professional even though in the last couple of days we had to face the realism of rainy weather and reluctantly move the production indoors instead of using our outdoor theatre area.
As part of the global campaign Send My Friend To School, the children made medals with a message which they wore during the opening ceremony of Dorelympics. The messages have now been sent to Nick Clegg to ask him to encourage other world leaders to reach the goal of getting every child into education by 2015.
Year 4 pupils enjoyed a fabulous day out at Hardwick Hall. Throughout the day the pupils extended their knowledge about The Tudors and they learnt all about the very rich lady who had the house designed and built, 'Bess of Hardwick'. The pupils were amazed by the number of windows that the Hall had and they understood why lots of people say, 'Hardwick Hall, more glass than wall'. The volunteers at Hardwick Hall provided fine details about how the house would have been in the Tudor period and provided the pupils with a real hands on experience. The pupils also had a volunteer to show them around the gardens. It was a delight to see them appreciating the smells and textures from the herbs and admiring the orchards. They found out lots about sustainability, and how it was possible for people to live off their own land.
Last Thursday, Year 1 went to Longshaw. The weather was thankfully beautiful, as we spent virtually all day outdoors! Y1S started with a "Gruffalo Walk", walking through the woods surrounding the Moorland Discovery Centre. The children learned about food chains and their fragility, camouflage and the water cycle before building some habitats for their teddy bears. All this whilst trying to work out who had been stealing the Mouse from the Gruffalo's nuts! In the afternoon they went on a minibeast hunt and were impressive bug-catchers! Mrs Longden's class were searching for minibeasts in the morning before they swapped over at lunch. Before we knew it, it was time to return to school and some children had been thinking so hard they fell asleep on the coach on the short journey! Many thanks to all the parents who offered to help with the visit and to all at Moorland for a lovely day enjoyed by children and staff!
Y1S children on the Gruffalo walk. Y1R/L hunting for minibeasts.
Building habitats for teddy bears. All food chains start with the Sun's energy.
Foraging for nuts like squirrels have to every winter. Now, can we remember where we left them? Y1R/L hunting for "worms". The brown ones were especially hard to spot. I wonder why...?
A particularly mini mini-beast! Taking a well earned break from building habitats.
51 Year Four pupils, enjoyed a 'WET' fun filled week at The Oakes!
The children enjoyed three days and two nights away on their residential. The rain did not prevent the pupils from taking part in a wide range of activities. Some pupils perfected their precision skills with a bow and arrow on the archery activity, whilst other pupils enjoyed the physical challenge of the climbing wall. Many brave pupils took on the Low Ropes challenge, completing a circuit of challenges off the ground, involving walking along a rope whilst their feet were submerged in very cold water and travelling at great speed down the zip wire. Laughing, screaming and shivering could be heard, as the children completed their Raft Session, travelling on a body board down a slide into the cold water. The warm showers were always appreciated! Between all of the fun, the children engaged themselves with the RE Christianity Curriculum, taught in an interactive way by the staff at the centre. All of the pupils were fantastically well behaved and were a great asset to Dore Primary School.
Congratulations to the loyal members of our cross country team for 2011-2012. We are a small but dedicated group of children and parents who have attended all the Sheffield Primary League races this season. We have had some excellent individual results - in fact Elliott Mckie in Y4 achieved 2nd place in Sheffield City in the League and in the Championships. What a fantastic achievement! Thank you to all our parents for their transport and support - to the DPA for providing us with a team flag ready for next season and of course to our resilient and courageous runners who show a marvellous team spirit :) We are looking forward to a celebration bowling event soon!
WE DO HOPE WE CAN ENCOURAGE MORE RUNNERS NEXT SEASON STARTING IN SEPTEMBER SO THAT WE CAN ENTER FULL TEAMS IN EACH RACE! Come on all you Marathon Club runners. We need you!


You've just completed a PT session and you've got one hour before your next session. You know you've got call-backs to do, paperwork to take care of or plans to implement.
You know you can get at least one of these things handled in the one-hour break…but then you start thinking…maybe I'll go for a juice, or check with trainers, or emails—anything rather than the thing your business requires.
Think about this for a moment. What happened to your original thought and intention: to act on a task required to create your business success? Where has your 'attention' moved to?
You thought about what you needed to do and then started thinking about doing something else.
You made a conscious decision to practise procrastination. Believe it or not procrastination is a choice; there are several reasons why at times we procrastinate.
Arousal types, or thrill-seekers who wait until the last minute for the euphoric rush.
Avoiders, who may be avoiding fear of failure or even fear of success, but in either case, are very concerned with what others think of them; they'd rather have others think they lack effort than ability.
For us to self-regulate and win by taking action, we need to know which type of procrastinator we are—then we can build a plan around breaking the habitual patterns of thinking and feeling that lead to procrastination.
Begin by making small decisions, and notice what you think and how you feel when the decisions you made generated the desired outcome.
Notice what you think and how you feel when the outcome is not what you expected to achieve. The key is learning to adjust and getting comfortable with making mistakes. You'll soon learn what does and doesn't work. "Even a mistake may turn out to be the one thing necessary to a worthwhile achievement" (Henry Ford).
Remember mistakes can be a natural occurrence when making decisions you've not made before. The trick is to focus on your WINS.
Remind yourself of all the times you've made the right decision.
Keep a journal of your progress and read it regularly.
Set a meaningful goal rather than focusing on ways to stop procrastinating.
Limit your choices by refraining from information overload.
Refrain from saying "I work better under pressure". Instead say, "I can do a much better job when I take time to complete the task at hand".
Choose to ignore feelings and thoughts such as, "I'll feel more like it tomorrow" and replace it with, "I can do it now".
Choose to begin the task, even if you don't complete it immediately. Give yourself a pat on the back for beginning.
Commit to daily meditation, even if it's for five minutes, or listen to calming music.
Reward yourself when you complete your task.
Refrain from judging yourself using negative self-talk by using an inner dialogue you'd use with your best friend.
Visualise for a few minutes the person you're becoming.
Work on self-acceptance by remembering your qualities and strengths.
Learn to recognise when you're avoiding doing something and understand that it's more than likely fear—false emotions that appear real.
Focus your thoughts on how you'll feel once you've completed your task. "The search for the perfect venture can turn into procrastination. Your idea may or may not have merit. The key is to get started" (unknown).
This article was written by guest author Lauren McLaughlin and originally appeared on the Create PT Wealth blog.


Matchroom, Crolla's promoters, shared the image of the Mancunian and the Ukrainian speaking backstage, Crolla with a towel around his waist and Lomachenko in his branded gear.
It was captioned 'Respect' - however there was no question that in the ring there would only ever be one winner.
Crolla told Sky Sports: 'I'm a bit gutted. I lost to a special fighter, I just wanted to give a better account of myself, but he hit me with a great shot. It's the first time I've been hit like that. As I went down I knew as frustrating as it was, I wasn't going to beat the 10 count, my senses went everywhere.
'I'm just gutted, I got in the best shape of my life and for it to end like that and as early as it did, very gutting.
'I don't know, I'm going to have some time to think about it. I wouldn't really want to go out like that but I've banged on about staying in boxing too long.


Presenting "what's happening in the forums" as it becomes a weekly (sometimes weekend) staple on a random day during the week. We're bringing you all the fun that happens inside the TmoNews forums!
How about that Fender edition MyTouch 1.2 with Eric Clapton? Chalk up another celeb endorsement from T-Mobile to help bring in the customers. Perhaps you're still looking for info on that T-Mobile 7.2 Mbps rollout? Other people are looking too; join them in your hunt for faster speeds. How about the Nexus One upgrade change/refund showing Google is actually taking customer complaints seriously and moving swiftly to rectify them. One of our exclusive stories this week was the rumor of a MyTouch "Slide" coming out. Though not everyone thinks it's such a hot idea, do you? Lots of buzz this week in my in box surrounding the sudden disappearance of the Touch Pro2 from the T-Mobile website, don't worry, we didn't think anything of it. Of course, TouchPro2 fans everywhere should be ready for the Windows Mobile 6.5 update next week bringing all sorts of goodies. That's all for now, unless of course you're a Modern Warfare 2 fan, then you should check out this thread!
Here is today's best news though. The good folks and friends of ours over at Phonedog have a "Pantech Blitz" going on right now for their One-Paw Bandit. They've asked us to help spread the word. It's not a T-Mobile phone you say? I know but this is how I see it: win it, sell it, and put the money toward a Nexus One/HTC HD2. Simple enough right? Show some love to these guys and play the game, they've been among our favorite sites for some time!
← "Hot" Handsets For T-Mobile's First Quarter?
Wow, I never thought I'd be the first post. Anyway, that's a good idea Tmonews.
I'm a huge fan of Wilma Flintstone. I was glued to the TV! And speaking of which, I just learned that there's a Wilma Flintstone song. Just downloaded it and quite a good tune to jam with while reading articles.
Wow, after looking at what a Pantech Blitz was, I'd definitely sell it. YUCK!!!
By all means sell it, easy money to put toward something else!
i thought it was the pantech matrix pro? not the blitz? oh well, i still played and lost.


text:
Q: Angular 2 Routing issue, No Output I am Learning Angular and am fairly new. I'm currently working on a project and just finished setting up my routing but now I am not able to see any of the content on the web page that I was able to before when the content was located in the app.component.html file.
It was then moved to a template in the buttons.component.ts file.
Can someone please tell me why there is no output?
app.module.ts
import { BrowserModule } from '@angular/platform-browser';
import {BrowserAnimationsModule} from '@angular/platform-browser/animations';
import { NgModule } from '@angular/core';

import {Routes, RouterModule} from '@angular/router';

import { AppComponent } from './app.component';

const routes: Routes= [
  {path: 'demo', loadChildren: './demo/demo.module#DemoModule'},
  {path: '**', redirectTo: 'demo'}
]

@NgModule({
  declarations: [
    AppComponent
  ],
  imports: [
    BrowserModule,
    BrowserAnimationsModule,
    RouterModule.forRoot(routes),
  ],
  providers: [],
  bootstrap: [AppComponent]
})
export class AppModule { }

---- demo.module.ts
import { NgModule } from '@angular/core';
import { CommonModule } from '@angular/common';

import { MaterialModule } from '../shared/material.module';
import { FormsModule } from '@angular/forms';

import { DemoRoutingModule } from './demo-routing.module';
import { ButtonsComponent } from './buttons/buttons.component';
import { FlexboxComponent } from './flexbox/flexbox.component';

@NgModule({
  imports: [
    CommonModule,
    DemoRoutingModule,
    FormsModule,
    MaterialModule
  ],
  declarations: [ButtonsComponent, FlexboxComponent]
})
export class DemoModule { }

---- demo.routing.module.ts
import { NgModule } from '@angular/core';
import { Routes, RouterModule } from '@angular/router';
import {ButtonsComponent} from './buttons/buttons.component';
import {FlexboxComponent} from './flexbox/flexbox.component';

const routes: Routes = [
    {path: 'buttons', component: ButtonsComponent},
    {path: 'flexbox', component: FlexboxComponent},
    { path: '**', redirectTo: 'buttons'}
];

@NgModule({
  imports: [RouterModule.forChild(routes)],
  exports: [RouterModule]
})
export class DemoRoutingModule { }

----- buttons.component.ts
import { Component, OnInit } from '@angular/core';

@Component({
  selector: 'app-buttons',
  template: `
  <button mat-button>
     <mat-icon>face</mat-icon>
     Click me!
  </button>
<mat-checkbox>Check me!</mat-checkbox>
  `,
  styles: []
})
export class ButtonsComponent {

}

redpajama_set_name: RedPajamaStackExchange


text:
<plugin>
  <name>app</name>
  <description>Elements used to communicate with applications</description>
  <filename>../../gst/app/.libs/libgstapp.so</filename>
  <basename>libgstapp.so</basename>
  <version>1.16.2</version>
  <license>LGPL</license>
  <source>gst-plugins-base</source>
  <package>GStreamer Base Plug-ins source release</package>
  <origin>Unknown package origin</origin>
  <elements>
    <element>
      <name>appsink</name>
      <longname>AppSink</longname>
      <class>Generic/Sink</class>
      <description>Allow the application to get access to raw buffer</description>
      <author>David Schleef <ds@schleef.org>, Wim Taymans <wim.taymans@gmail.com></author>
      <pads>
        <caps>
          <name>sink</name>
          <direction>sink</direction>
          <presence>always</presence>
          ANY
        </caps>
      </pads>
    </element>
    <element>
      <name>appsrc</name>
      <longname>AppSrc</longname>
      <class>Generic/Source</class>
      <description>Allow the application to feed buffers to a pipeline</description>
      <author>David Schleef <ds@schleef.org>, Wim Taymans <wim.taymans@gmail.com></author>
      <pads>
        <caps>
          <name>src</name>
          <direction>source</direction>
          <presence>always</presence>
          ANY
        </caps>
      </pads>
    </element>
  </elements>
</plugin>

redpajama_set_name: RedPajamaGithub


Len is self employed as a computer consultant.
He is a member of Lodges Piako 160, Te Aroha No 52, and Waikato Lodge of Research No 455. Royal Arch Chapters Piako No 48, Ohinemuri No 17 and Research Chapter No 93. Waikato Cryptic Council No 22. Secret Monitor Conclaves Piako No 43, Waikato No 8, and Three Arrows Research No 30. Hauraki Rose Croix No 1063. Waikato Red Cross of Constantine No 8.
He is Married to Jacqueline, and have two sons, one living in Morrinsville and one in Colorado USA. With two Grandchildren in NZ and six in USA.
Len lists his interests as photography, car racing especially Australian Super V8s and of course Computers which he made into a business 26 years ago.
Murray is a retired Electrician / Engineering manager.
He did his schooling at Ohura, Wanganui Boys College and Papakura High School and then went to Auckland Technical Institute completing his Electrical Apprenticeship in 1969. Most of working life has been spent in the Waikato.
He was initiated into Lodge Hillcrest 363 in November 1994 and is currently the IPM having also been Master in 2001. Murray has spent a lot of his Masonic career trying to update and bring his Lodge into the 21st Century. He has held a collared position every year since becoming a MM.
Murray lives in Hamilton married to Iris. They have three children and seven Grandchildren. A lot of his spare time is spent with family.
David has a musical Instrument retail store in Hamilton which was started by his late father Andy Shearer (Past Grand Organist) in 1963.
He was proposed into Lodge Tawhiri 166 by his father and is also a companion of The Tawhiri Royal Arch Chapter No. 97.
He is married to Susan and they have three children aged 20 , 19 and 16 years.
He is involved in a number of community based services and enjoys giving back to the community both he and his family have grown up in.
His interests are music, fishing and golf and spending time with family and friends.
Don joined Lodge TeMarama No. 186 with his Brother Stan in a double 1st degree when his Father, Murray Seath was Master in 1971.
Don is married to Susan and together they have 3 adult children. They are all married and have 3 children each, making Don and Susan the proud Grandparents of 9 Grandchildren. Don's Family are the most important part of his busy life.
Don is a retired Dairy Farmer who has held various Directorships in Agriculture related Companies. He is a Chartered Member of the Institute of Directors.
He has been an active contributor in his community, with roles in Scouting, Farm Cadet Scheme, Schools, Rowing, Club Rugby Conservation, and currently the Waikato-Hauraki-Coromandel Rural Support Trust. His favourite past times are sailing and travelling.
Don has gained huge benefits from Freemasonry in the influence it has had in his life. He is keen to use the skills he has gained to help the Waikato District adjust to the changing needs of future Freemasons.


text:
The Politics of Oxygen
Today's WSJ has a fun profile of Peter Orszag, the Director of the Office of Management and Budget, and the challenges he faces making cost control a key part of health care reform.
I particularly enjoyed this episode:
The battle heated up in June, when Mr. Orszag visited Capitol Hill to discuss health care with a small group of House Democrats. The meeting started well, with one lawmaker after another echoing his message that spending controls were critical to any health-care overhaul, according to two administration officials.
Then one member said her top priority was winning higher payments for oxygen suppliers, the officials say. Mr. Orszag was taken aback. Officials had been trying for years to cut payments to suppliers of oxygen and other medical equipment, which critics say are inflated. Yet when a new competitive bidding process was set to take effect last year, industry supporters in Congress were able to delay the plan. They are still fighting to block changes.
"One of the reasons we currently have such disjointed and skewed incentives is that we have an excessively political process," Mr. Orszag said in an interview.
I think Peter is absolutely correct.
When I first worked for Congress, I was stunned by the amount of time and effort that members gave to issues that struck me as minutiae. This was particularly severe in health care. Congressional staff — and, at times the members themselves — would worry about things like payment rates for wheel chairs, bidding rules for oxygen suppliers, and other micro-health financing issues.
Those micro issues are, of course, a big deal for the individuals involved. And it's natural that they would go to Congress to express their views, since Congress ultimately controls the payment rates for programs like Medicare.
But it still strikes me as a fundamental mismatch between what Congress ought to be doing — establishing the broad policies that guide the delivery and financing of health care — and what Congress actually does.
As the health debate moves forward, it would be useful to consider whether some decisions might best be transferred from Congress to other decision makers. At Peter's initiative, the Obama administration has offered one approach: empowering in the Medicare Payment Advisory Commission to make some decisions about payment rates. MedPACs decisions would go into effect unless Congress over-ruled them. That still leaves some power in Congress's hands, but perhaps it will shield the payment process from so much politics.
A bolder approach, well beyond the bounds of the current debate, would be to move Medicare to a premium support model, in which the government provides subsidies for seniors to purchase insurance from competing providers (somewhat similar to what happens in the current drug benefit). Premium support raises a host of challenges. But, at least in principle, it would provide a way to focus Congress on big picture issues — what's appropriate minimum coverage for seniors, how much should taxpayers chip in, etc. — while leaving the micro issues to the competing insurance companies.
My point is not to recommend either of these approaches specifically, but just to suggest that it would be useful to consider health financing reforms that would separate Congress (and, therefore, politics) from individual payment decisions.
Author DonaldPosted on July 24, 2009 July 24, 2009 Categories Budget, Health, Politics
3 thoughts on "The Politics of Oxygen"
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While I was at OMB, I once joined the Director at briefing for congressmen. During the Q&A they gave him a lot of grief that the Administration wasn't doing enough to reduce spending. One member, in particular, stood up and gave a dramatic performance. Afterwards, as the meeting broke up, that very same member came over and quietly asked the Director to support a water project in his district. Some things never change.
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http://www.mwctoys.com/REVIEW_060204a.htm). It was then that I decided that no matter what, it would come the day I would create a Birdman custom.
Typically I start by looking if there are alredy previous custom attempts before starting mine. The odd thing is that I've seen lots of Space Ghost customs but almost no Birdman customs (I just have seen one, will not say whose, which showed me exactly what NOT to do).
So then I had to search for photos and production model sheets to get a better idea of the proportions, the helmet and such. Some shots from the Toynami 6" figure and, specially, from Toynami's maquette ( http://www.toynami.com/popup_birdman07.html ) which also helped me to see the kind of orange and blue tones that would work better. Then the second thing was to find the proper base figure for it. The wings were a no-brainer: JLU Hawkgirl or JLU Winged Amazo were good options. Actually I had a Winged Amazo lying there in the box, but the body lacked the articulation I wanted for the figure. So finally I choose a 10POA Mattel JLU John Stewart as basis. But if you do not mind articulation, go for the Winged Amazo or the JLU Hawkman, it will save you loads of time and head-aches. For the head, after some search in the box I saw that the Mattel Secret Wars Captain America head had the right jaw line and a bit of a goofy look, ideal for the character.
One problem I faced with the chosen base figure was the lack of a waist line thick enough to place Birdman's belt. So I had to crack the body open to try to find a solution for that. Also I had obviously to create a hole in the back to stick the wings in. In the image below you can see the starting point, with the body already cracked open.
I had to adapt the waist axis by re-sculpting it so I could create a gap between the upper and the lower body. I filled-in the gap space with two pieces of laminated wood, which were then hidden by the belt (made of paper, with the recipe for belts I explained in the previous post). Unfortunatelly I have no photos of this step as sometimes my urge to advance in the custom work makes me forget on documenting the process. I also started to sculpt part of the helmet detail directly in Birdman's head. I kept part of Captain America's head wings to become the reinforcement of the mask flaps. And I sanded down Green Lantern's chest and upper arm costume lines.
It was then when saw that the white in the wings did not worked ok with the rest of the figure. So I went again to check Toynami figures to see which hue of blue was used instead. When I re-painted the wings with a clear blue then I confirmed that was the right color.
Another character I loved as a kid was Black Vulcan. Again here we have a character with a lame performance in the Super Friends show that strangely enough do have fond memories of. I was dreaming to have a Black Vulcan 5" figure for a long time, since I had managed to get a Kenner Superpowers Samurai figure in eBay. And I had for more than one year already one Mattel JLU John Stewart figure reserved for this custom, but would never start the custom. But my interest came in again with AFI's JLU custom showcase announcement, and this was the trigger for me to finally go for it.
Again the first step was to search for photos and production model sheets to get a better idea of the proportions of the chest pattern, the mask and the ear boomerangs. This time I was shocked with the great amount of Black Vulcan customs I found on the internet. From these I learned several things, such as that the chest bolt patterns are critical to make the figure believable, so they had to be in a good proportion with the body. I went several times to the original Alex Toth model sheets to try to determine the bolts' proportions and how to adapt them to the wider JLU body. The second thing I learned is that black looks better than many blues. So it was my personal decision to use black instead of dark blue for the figure. Special care was put into the ear pieces, which are more model-accurate than the ones seen in many customs out there and Mattel's prototype.
Although is not a very original or difficult custom, I'm very proud of the final look of it.
One nice thing is to see how it looks close to my Kenner Superpowers Samurai Figure. Although it is clear the distinct sleek look of it, Black Vulcan's figure is in scale with Samurai.
And to close the post today, just a quick comparison between Birdman and Black Vulcan, showing how the same body can give as result two totally different customs. Also it can be seen the extra height in the case of Birdman due to the waist gap for the belt.
In the next post I will reveal the recipe for my Starfire custom, plus I will show two extra female customs I've made recently.
Last edited by mentalpower on Sat Jul 11, 2009 3:50 am, edited 1 time in total.
As I explained in my first post one of my earliest customs was Halo. Since the very beginning the second female custom I wished to do (and never could) was Princess Koriand'r, better known as Starfire. I have been years and years dreaming of doing a custom of the character, but faced a problem: how to sculpt all that hair! The solution came when I got tired of my DC Direct Teen Titans PVC set, and noticed that the hair of the Starfire PVC figure in the set was a bit too big for that figure, and thus might be the right size for a 5" custom.
The body to use was cristal clear to me years ago when I'd bought the Toy Biz Alpha Flight Vindicator Figure, as it had the right... ehem! proportions and curves for Starfire. Therefore I had already one Vindicator figure reserved for this figure in my spare parts box.
a Toy Biz Marvel She-Force Dazzler was a good match. Then I used a drill to create the hole in the hair piece for the Dazzler head. The resulting base figure can be seen in the following picture.
In the image you can se that I also sketched the lines for the suit. I typically do not do this, as the final decision on where each line will go is made directly in the final phases of the painting. But this time, after looking to the Mattel DCUC Starfire figure, I saw that just painting the costume on the body would not work, as Starfire is not wearing a costume but a metalic armor. This all meant that I had to sculpt some of the armor lines in the figure, not jsut paint them, and to ensure that they would look right I needed to sketch them first.
The most time-intensive part of this custom was sculpting the armor. It took actually several afternoons after dinner to do it. All the armor (except for the collar, the wrist bands and the boots' vertical ornament) was carefully sculpted down with a x-acto knife and sanding paper. The collar, wrist bands and bracelet were made with sanding paper (to get the textured look) plus regular paper hardened with superglue. The boots vertical ornament is made of a total of 8 paper pieces, all glued together and hardened also with superglue.
Once all this was added to the figure, I made a first coat of paint to check the overall look and decide the next steps. You can see it in the following image.
For the armor I prepared a metallic light purple by mixing roughly 3/4 silver paint with 1/4 purple paint... The belt bucke comes from the PVC figure itself, but the collar pendant was hand-made in paper.
Apart from Halo, a member of the Outsiders that I liked a lot was Looker, in her original look (way before they converted her in a strange mutant vampire with a horrible costume). The choice for a base figure was easy here: it is a Toy Biz Marvel She-force Miss Marvel (which is actually a straight repaint made by Toy Biz from their X-Men Phoenix figure). The only tricky part of this custom was to eliminate the belt by carefully sanding it down with an x-acto knife and sanding paper. Then the different details were added using harded paper. The cape is not paper, but made of a normal white plastic bag (from a comic store in Barcelona), and the golden chain was from a old pen that had a chain to connect it to its base. Some time was also spent to repaint the whole face, including the eyes, eyelids and make-up. The result can be seen in the image below.
Another DC superheroine I liked was Power Girl. Some of you may say I'm crazy because I like her second JLE costume... but I really do! So her look in the JLE comics was the one I tried to reproduce in the figure. The base figure is clearly a Toy Biz X-Men Secret Weapons Force Jean Grey with bracelets, collar, earrings and buckle made of superglued paper. The result is a not too difficult but effective custom, in my opinion.
Although I'm quite a DC fan, I also do some Marvel characters, specially when there is a clear gap in my collection (or to avoid paying huge amounts of money for an exclusive figure that is just a repaint, but I will talk about this in another post). The following are some recent customs of some femal Marvel characters.
Mary Jane Watson (also known as MJ)... years and years waiting for a cool figure to go with my 5" Multijointed Spiderman (I'm not counting the straight, horrible repaint Toy-Biz made from a Invisible Woman figure in yellow and blue, or the even worse Santa-clad version of it for the Christmas two-pack) and the perfect figure never came. Then, after years of 6" Marvel figures, I was excited with the announcement of the recent 5" figure inspired in the Spiderman 3. It looked great on pictures, so I decided to buy one through BBTS. But I was really dissapointed when I finally received the package and saw the figure. First it was totally out-of-scale in comparison to my 5" Spiderman figure, but worse than that Mary Jane's body was not what I was expecting for a beauty, well-proportioned fashion model. And that dress!! Is that fashion? The guy that created this figure has a strange concept of female anatomy and fashion, to say it in a polite way.
So then the task was to find a better, nicer body with also a cooler clothing, closer to what Mary Jane would wear. And I finally found the perfect match in the Underground Toys Torchwood line: Gwen Cooper. So the task here was to add the Mary Jane head to the Gwen Cooper body. Unfortunatelly I have no photos of the process. Gwen's head has no articulation and is actually part of the torso, while MJ's head has a ball joint that I wanted to keep for the custom. So what I did was to cut the neck in both figures to almost the same height, then adapt Mary Jane's neck to Gwen's body (it was actually thicker, so I had to sculp it a bit. Then I glued it and hid the joint by carefully sanding it down. The final touch was to paint the neck so the joint is unnoticeable. Finally after looking into some classic images of the character I decided to paint the pants in a light brown color.
In the next shot we can see the comparison between Mary Jane's old, skinny, hunched body (with Gwen's head just added here for the comparison) and the new one. What do you think?
And just to end with Mary Jane, a scale comparison with other Toy Biz 5" figures below.
Although it is a bit taller that earlier Toy Biz 5" figures, it is quite in scale with the later ones.
was hoping that they would eventually produce a 5" Crystal figure, but never happened. Then I saved a Toy Biz Fantastic Four Invisible Woman figure to make one myself, but the project was left for years. And it didn't came to my mind until I had the Torchwood Gwen Cooper's head (left from the MJ custom above) in my hands and I was wondering what could I do with it. And then I realised it would be great for Crystal.
This custom was not difficult. I spent most of the time browsing several pictures from Crystal to choose which, from all the costumes, I would use for the custom. Finally I decided to go for a very recent one.
Then I created the few costume additions in hardened paper, including the head ornament, and added several coats of paint covered with glossy or matt varnish. I must say I'm happy with the result.
And in the next post I will reveal the entries I was preparing for the JLU CUSTOM SHOWCASE some months ago but failed to deliver on time. So stay tuned!
Last edited by mentalpower on Thu Jul 16, 2009 9:05 am, edited 2 times in total.
Ok, so in this post I will show you some of the customs I was preparing for last year's JLU CUSTOM CHALLENGE. I was really happy with the idea when it was posted as I was starting my own JLU customs and the showcase was just the perfect excuse to push myself to finalise some of the customs I had just started or was planning to do. So I started asking lots of questions about the rules that Superfly and others patiently answered. Unfortunatelly when the rules were finally revealed I was too busy at work, and the customs I had almost finished were not fully following the rules: to be only repaints and use only Mattel JLU parts.
As I think there are some good ideas there I decided to post them now. I'll try to explain for each one how I did the custom, but also how the custom can be simplified to make it feasible for Mattel to do it at a reasonable price.
So today let me present part 1 of these customs (sorry, but the amount of work these days makes that some of the photos I've taken have not been processed yet, so this will be a three-part post).
This custom and the next are Flash Rogues, as I felt that they are underrepresented in JLU. In this case is thr Weather Wizard, but not in its animated version (that is, his appearence inthe superman Animated series) but using his current look in the comics.
The base figure is as JLU Kyle Rainer. The bands on the chest are made of paper. The collar is a piece cut from another figure's cape and the wand is a real pin, with the tip slighly trimmed so it would not be that dangerous (but of course is not adequate for kids).
The same figure can be made without the paper add-ons, eliminating the collar (just painting it on the neck) and changing the dangerous metal wand by a soft, kids-safe one.
This one came to be trying to think what to do with a JLU Luthor I had in my box. And seeing the face the first character that came to my mind was Heatwave (which is odd: seeing some people's customs of Vandal Savage using the same figure I still wonder why I thought about Heatwave instead).
Here I'm using again the modern look of the character, not the animated one, as I did not liked itls look in the JLU show. I've seen a custom made by shazam37 which uses a similar look, and one by Scott Rogers in AFI's custom Showcase that used an even whiter, more classic look.
Basically I kept the belt from Luthor, sculpted the top of the gloves directly in the plastic with an x-acto knife, and adapted the gun holster to go only over one shoulder. I created the shoulder pads in paper (then hardened with super-glue), added the gun holster and the thin costume strips. The googles are made of two pieces of clear plastic coming from a JLU bubble. Finally I painted it all and sealed it with matt varnish.
The same custom can be made with a regular Nemesis or Luthor gun holster and the costume strips and soulder pads painted on. The googles will be a problem, though.
The custom made by Scott Rogers (based on the Mirror Master figure) is easier to make than mine and has a more classic look.
I wanted to give it a more distinct look compared to the regular JLU Hawk Figure and thus did extra efforts to make it have unique features. One of the things I'm not happy with the animated look of Hawh is the over-simplification of the cape. For Kestrel I wanted to recover a bit more the look of multiple feathered cape that had the character designs in the comic, so in practice I wanted to find a way to extend the two unconnected cape feathers on the back of the figure to the front. I did several skethes with paper pieces of different shapes until I found the shape that gave it the right look.
Also I changed the head to one less serious, one that I could give a more evil look. I had not many options in my fodder so I had to go for a JLU John Stewart head which I then sculpted a bit to reduce the prominent lips. To adapt the head to the body I cracked open the body, sealed it back with superglue and then had to re-shape elbows to adapt them to a narrower neck. I also sculpted a bit the space between the two cape feathers on the back of the figure, to make them look like they were not connected.
Once all this was done I added the feather extensions to the cape, made from thin carboard hardenend with superglue, and painted it all. To increase the contrast between the black and the purple, I varnished the black parts with glossy paint and the purple parts with matt varnish. The cowl black stripes are cut from a plastic-coated wire tipically in computer wires packaging and can be placed in different shapes. And the result is shown below.
To better see how distinct is from the original figure, in the images below I show Kestrel side by side with the JLU Hawk figure.
The same figure can be made with the JLU Hawk body and a Mirror Master head with no add-ons, closer to a custom I've seen recently (but cannot rmemeber the customizer, pelase accept my appologies). The trick to keep some distinct look would be to keep the proposed painting scheme, specially to paint black the top half of the figure.
Okay, that is for today. In the next days I'll post part two, with some goodies to keep the balance between good and evil.
Ok, so in last post we so some baddies' customs, so in this one I will show some super-hero customs I had planned for the JLU CUSTOM SHOWCASE.
The release by Mattel of the JLU Obsidian figure was clearly demanding for her sister to be made in plastic. I've seen two or three Jade customs, using different JLU heads as basis. But in my case Jade was already a planned custom years before the JLU line, and I had already picked the perfect head for this: Trixie from the Resaurus Speed Racer line. My original plans were to use a Toy Biz Invisible Woman body, which I already adapted for the head, but after doing a first paint shot I saw the proportions were not right. So the custom was laying in my box for years until I saw the potential to use the head in a JLU body (the head-less Invisible Woman body was recently used in the Crystal custom I've shown some posts ago).
The trickiest part of this custom was to crack the body of a JLU Justice Lords Wonder Woman to adapt it for the new head, and it was tricky because JLU female bodies are very fragile. Once I had adapted the neck hole to the new head the second trickiest thing was to seal the body with superglue without the head and the limbs being stuck. Once this was done it was a matter of sanding down imperfections and paint the whole figure.
In this case I had a body (TNBA Riddler) waiting for the proper head and for a solution to the cloak. Some years ago I solved the latter, with a cape coming from Toy Biz Most Wanted Blink. And roughly a year ago I'd found the proper head: Lex Luthor from the Dc Direct The Death of Superman line.
So having all the pieces together the trickiest part was to adapt the head to the body. As both the Luthor head and the Riddler body had peg holes but no peg I decided to loose the neck articulation... so I glued the head to the body and sculpted the joint in a way that would then dissapear when painted.
I added then the goves and boot tops, made from very thin paper hardened with superglue, and painted the figure. The cloak was then glued directly to the figure in three points; two in the upper chest and one in the head.
The Luthor head is from a DC Direct figure that may be off-limits to you. The Deadman's head is a good alternative, just with a different shadow arround his eyes. The cloak may require some work from your side, though.
This was not part of my planned JLU customs for the showcase but it is fresh from this past weekend so I cannot resist to share it with you. As you may have seem The Titans is one of my favourite teams and it is well represented in my customs (Starfire, Changeling, Troia, and more that i'll show in future posts). So this time spotlight is on Toni Monetti, better known as Argent.
The original hair was taken apart, the head band was sanded down and the belly was sculpted. Wrist bracelets are made from regular paper hardened with super-glue (as usual in my customs). The boots' ornaments and the belly piercing are made from a thin cardboard (shown in the image above). Actually I added this cardboard into the shot so you can get a better idea of my technique: first I made simple patterns for the boot shapes using regular paper, and when I have the right shapes I copy those shapes in the cardboard. In this case I glued the original paper patterns into the figure to serve as guidelines for glueing the boots' ornament pieces in the right spot. Also in the cardboard we can see a donut-shaped pattern that corresponds to the round collar for the Birdman figure, and the feather-shaped pattern is the one from the Kestrel cape feather add-ons.
Okay, that is for today. In the next days I'll post part three, with few off-the limits figures made also in JLU style.
The quality of your customs have improved significantly!
The 'fun character choices'... well, some of it is really driven by my interest in a particular character never made in plastic before, and sometimes is inspired by the figure itself (is what I call 'opportunistic customs', Kestrel is an example). And sometimes, as when SDCC is approaching, I tipically go for obscure characters I think no-one will ever do (but then I was wrong with, e.g., Black Vulcan) so no matter how obscure the character is it may get into plastic in the end.
I am completely impressed. EXCELLENT!
Thanks dude! I really liked your Donna Troi and Power Girl entries for the JLU Custom Sohowcase. And must admit I might take your recipe for a Vandal Savage figure... (if Mattel shows no plans to make it themselves).


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Q: Can't locate IO/Pty.pm - but it is installed I have a script that I inherited from another developer and am trying to make it work on a new RHEL server.  The script uses Expect.pm which 'uses' IO/Pty (and IO/Tty).
The issue is, when I run this script, it claims it cannot locate IO/Pty.pm.  The path to both IO modules is /usr/local/lib64/perl5/IO. This path is in my @INC.
I have verified the permissions match what other functioning modules have (also tried 775 as a test).
The only difference on the new server vs old is the version of Expect.pm and IO/Pty and IO/Tty (newer versions on the new server).
Any idea how I can get this to work?
Here is the error:
[Tue Dec 17 14:33:51.284748 2019] [cgi:error] [pid 64712] [client redacted:63776] AH01215: Can't locate IO/Pty.pm in @INC (@INC contains: /usr/local/lib64/perl5 /usr/local/share/perl5 /usr/lib64/perl5/vendor_perl /usr/share/perl5/vendor_perl /usr/lib64/perl5 /usr/share/perl5 .) at /usr/local/share/perl5/Expect.pm line 23

drwxr-xr-x. 17 root root 4096 May  8  2019 /usr
drwxr-xr-x. 18 root root 4096 May 20  2019 /usr/local
drwxr-xr-x.  3 root root 4096 Jul 12 13:44 /usr/local/lib64
drwxr-x---   4 root root 4096 Dec 17 12:18 /usr/local/lib64/perl5
drwxr-x---   4 root root 4096 Dec 17 12:18 /usr/local/lib64/perl5/
drwxrwxr-x   3 root root 4096 Dec 17 14:56 /usr/local/lib64/perl5/IO
-rwxrwxr-x   1 root root 9114 Sep 12  2014 /usr/local/lib64/perl5/IO/Pty.pm
-rwxrwxr-x   1 root root 8096 Sep 12  2014 /usr/local/lib64/perl5/IO/Tty.pm

A: Your application almost certainly doesn't have access to one of the intermediate directories:

drwxr-x---   4 root root 4096 Dec 17 12:18 /usr/local/lib64/perl5

You can fix this (as root) with chmod o+rx /usr/local/lib64/perl5


4 million people worked from home in 2012, with a 13% increase in the last 5 years. What employees used to see as a once a week perk in companies that had the infrastructure to support it, has rapidly turned into a fast growing trend. And it shows little sign of stopping.
Contributing factors to this include significant advances in affordable technology, faster broadband and cloud based services. In a time of double and triple dip recessions, employers are looking at ways to cut costs. This can be by outsourcing or allowing staff to work from home, either way the savings can be considerable.
– Real flexibility: allowing people to work to their own timetable. Get up early, stay up late or work weekends, just as long as timescales are met.
– Money and time: not having to travel to work saves both, as well as benefiting the environment.
– Increased productivity: no noise or distractions from working in an office atmosphere means better concentration and ultimately more achieved.
We're big advocates of agile working at Platform Twenty, and many businesses are catching on to the benefits of remote workers. You can hire the best talent to work on your project (regardless of location), overhead expenses can be reduced dramatically (especially if you downsize the office space or get rid of it completely) and studies have shown it can also improve productivity.
However we're also aware of its potential drawbacks; people may feel isolated, cut off from colleagues, miss the ability to bounce ideas off each other, and find it hard to stay focused.
Good communication is the key to making remote working work! Team members need to be able to collaborate easily by sharing resources and ideas quickly and effectively. Luckily a vast array of online tools exist to help achieve this. Being the helpful people we are at Platform Twenty, we've put together a list of the ones we find most valuable.
We looked at cloud based storage and sharing apps Dropbox & Google Drive in our previous post. Both have strong collaborative benefits, as listed below. However a new contender in this field is BCSocial.
BCSocial provides a limitless cloud storage space. It can be accessed anywhere on any device and users can share, comment on and in, any content. They're claiming it will reduce the need for emails. We'll be reviewing it in more detail in a future article, it's definitely one to keep an eye on.
Dropbox Collaborative Benefits – First 2GB free (can be increased by referring friends to service and paid subscriptions offer up to 500GB of storage). Can set up access for shared folders, making it easy and secure to share files with remote colleagues. Dropbox for Business (1,00GB of storage) allows you to switch access from one user to another, great when outsourcing to different people for various projects.
Google Drive Collaborative Benefits – stores up to 15GB for free, comes with Google's search tools making it easy to find that allusive file a colleague didn't name correctly. Provides the ability to work live on documents with co-workers without ending up with multiple document versions.
Skype is a great collaborative tool for thoseworking remotely, allowing voice, video and instant messaging to other Skype users for free. As well as your home PC and laptop, it works on tablets and smart phones, perfect for when you're out and about.
Collaborative Benefits – at the end of last month Skype announced that their Group Video Calling (GVC) would be free of charge to users of Windows desktop, Mac and Xbox One, with other platforms following shortly. You can also have voice calls of up to 25 people. The messaging tool is as close to sticking your head up over the partition and asking your colleague a quick question as we can find.
Webex provides a platform for online meetings, web conferencing and videoconferencing. Meet online and share files, information, and expertise. WebEx is available across most devices including iPhone, BlackBerry and any Wi-Fi or 3G/4G-enabled mobile device.
Collaborative Benefits – we love screen sharing, it allows you to show colleagues and clients what you're working on & see their reaction in real time. Webex allows you to annotate when screen sharing, a great tool for highlighting exactly what you're talking about. Schedule invites and reminders so everyone turns up when they're meant to. Free for meetings with up to 3 users, then various packages available.
Basecamp is a great project management tool which allows you to start message threads, assign tasks and view work scheduled on a calendar. It also integrates with email and allows you to store related documents and files. Having been around for 15 years they know what they're doing and aren't about to disappear. Easy to use across various platforms, with apps for iPhone and Android users.
Collaborative Benefits – Easy to use interface. See how projects are progressing, check to-do lists and get notified when colleagues mark an item as done. All without the need for constant emails. Great for continuity when working across time zones. Free 60 day trial, and then packages starting at $20 a month for 10 projects and 3GB space. Can assign people to projects without them needing a paid account.
Trello is another a project management tool, potentially simpler to use then Basecamp and a big hit with fans of Kanban boards. We'll be looking at Trello versus Basecamp in a future post so don't forget to fill in your details on the right and we'll keep you updated.
A CRM system is an essential collaboration tool, providing a single view of the customer which everyone in the company can see and add to. Recent studies show companies with a fully utilized CRM system can increase sales by 29%. They provide important information for effective marketing campaigns, show the sales pipeline and help you make the most of customer feedback.
Our favourites are the self-hosted CRMs such as Zurmo, Sugar and V-Tiger. Choosing the right CRM needs a post all of its own, and we're not going to let you down! We'll be publishing one shortly so sign up and don't miss out (we hate spam and promise to only send you the good stuff)!
Inspired by the above but take it a step further? Check out Sqwiggle. The app shows constant updated stills from your co-worker's webcams. Easy to see if they're around, and by clicking the picture you can instantly chat to them. No calling, no accepting, just immediate communication.
We'd love to hear your remote working tips and learn about any awesome tools you use, so please share in the comments and get social with us on Twitter and Facebook.
In addition to Skype and Webex, you may try tools like on premise R-HUB web conferencing servers for collaborating online. It provides 6 real time collaboration applications in one box.


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A Summer of Success for Sage Intacct
By Brittany Benson, Senior Corporate Communications Manager at Sage Intacct | July 23, 2018
It's been a busy and successful summer here so far at Sage Intacct and we wanted to take a minute to update our readers on some fabulous awards that our organization and employees have recently received!
Sage Intacct's Tina Tung Named to Silicon Valley Business Journal's 2018 Woman of Influence List
Tina Tung, a Sage Intacct Release Manager, was named to the Silicon Valley Business Journal's 2018 Women of Influence List. The women named to this tenth annual list were selected from across Silicon Valley for their influence in their industries, communities, board affiliations, and other leadership roles.
Tina was named to the list for her leadership at Sage Intacct, overseeing software builds and releases from testing through deployment. Most notably, Tina developed and promoted the Sage Intacct Continuous Integration/Continuous Delivery system and has contributed to the development of Sage Intacct's artificial intelligence and machine learning business tools.
Beyond her incredible work at Sage Intacct, Tina also runs her own Baskin Robbins and pastry establishment. She also volunteers regularly with Loaves and Fishes and local Bay Area dog rescue organizations.
"The leadership, integrity, and hard work of Sage Intacct employees allows us to deliver high quality products and exceptional service for our clients," said Rob Reid, Managing Director and Executive Vice President of Sage Intacct. "Tina's attitude and tenacity has helped us build systems and processes that enable the delivery of quality releases quarter after quarter. We are proud to see Tina honored for her contributions to the success of our business and the success of the Silicon Valley community."
29 Sage Intacct Partners Named to Bob Scott Top 100 VARS List
Industry influencer Bob Scott recently released his Top 100 VARS list for 2018, including 29 Sage Intacct partners on the annual list.
The Bob Scott Top 100 VARs list recognizes the annual achievements and growth of mid-market accounting software Value Added Resellers. The 2018 ranking of the Top 100 firms is based on annual revenue and this year's list highlights how the shift to the cloud continues to change the market.
The Sage Intacct Partner Program has grown steadily since its inception in 2010, and continues to attract the industry's top organizations. Sage Intacct's network of partners deliver comprehensive, high quality services, support, and software to help companies get maximum value from their Intacct investment. Sage Intacct partners currently deliver nearly half of the new business Sage Intacct generates and are spread across all regions of the United States, as well as in Canada.
Sage Intacct Recognized as 2018 Bay Area Best Place to Work
Sage Intacct has been named to the 2018 list of Bay Area Best Places to Work awards program presented by the San Francisco Business Times and the Silicon Valley Business Journal.
Additionally, the Bay Area News Group has named Sage Intacct a winner of the Bay Area Top Workplaces Award for the 8th consecutive year.
Both award programs honor Bay Area companies with exceptional workplaces. The lists are comprised of a selection of companies that were highly ranked by employees, have fun and collaborative cultures, offer solid compensation and benefits, exhibit strong management practices and overall workplace satisfaction.
"Ensuring our employees are happy and engaged drives the overall business success of our company," said Rob Reid, Managing Director and Executive Vice President of Sage Intacct, "A healthy work environment where our employees can thrive powers the delivery of exceptional service and innovative products to our partners and customers."
This is only the latest of a number of workplace awards presented to Sage Intacct. The company was recently honored as one of the 2018 Best Places to Work, Bay Area by FORTUNE and Great Places to Work. Other honors include:
Best and Brightest Company to Work for in the Nation by NABR
Best Small & Medium Workplaces FORTUNE and Great Place to Work
Best Workplaces for Recent College Graduates
Best Place to Work in North America
San Francisco Bay Area's Best and Brightest Companies to Work For
To learn more about a career at Sage Intacct visit: https://www.sageintacct.com/careers.
Brittany Benson is the Senior Corporate Communications Manager at Sage Intacct.


"Let's walk! We'll get there faster on foot!" A cackle erupted from the wrinkled face of a woman peering down at us through thick glasses. Manuel and I nodded in agreement and exchanged bemused glances as the sharp-tongued Jamaican grandmother ambled off to join the crowds clustered around the information desk. We were sprawled out on an empty patch of carpet at a departure gate at Heathrow Airport, the very gate that was supposed to have been the embarkation point for Air Jamaica Flight JM002, 19 hours earlier. Around us families had staked out little patches of floor for themselves, napping, snacking and stopping airline employees to harass them for updates. Children careened around this human obstacle course, diving under chairs and tumbling into strangers' laps. Young Jamaican women in tight skirts and impossibly high heels (how do you survive a long flight in those?) strutted up and down the rows of people, deliberately catching the eye of every man under 80, while plump grandmothers clucked and shook their heads disapprovingly. This was human theater at its finest, the spectacle suspending itself at periodic intervals so that the tinny voice of the departure gate agent could be heard expressing regret at the latest extension to the flight delay. From the looks of it, we weren't going anywhere soon, but at least we weren't starved for entertainment.
We had arrived at Heathrow the previous afternoon in plenty of time to make our connection to Montego Bay. A one-hour flight from Edinburgh and the ten hour flight to Jamaica had been supposed to deposit us at our destination by early evening. We would have a driver meetings us there to take us down to the South coast, where hopefully a shower, a late dinner and a relaxing night in a room directly on the beach were waiting. But on our arrival at Heathrow we had been informed that due to mechanical faults, one of the two Air Jamaica planes used to fly to London was out of service, and our flight had been postponed until the next morning. We were handed a hotel voucher, free dinner and breakfast slips for the hotel restaurant, and the news that we would be picked up at 6am sharp for a 7am departure. We were dismayed, but not crushed – by our calculations we would be arriving early the next afternoon and still have the better part of a Caribbean day to soothe our delay-frazzled nerves. I just hoped our driver would stick around.
After dumping our bags at the airport hotel, we assessed the situation. We were in London for a night, we weren't paying for it, and there were dozens of restaurants a short tube ride away that I was dying to try. "We're not going to eat dinner in the hotel, are we?" Manuel asked, eyeing the vouchers warily. I laughed and shook my head. "Okay, but do you have any specific place in mind?" Surprised by my own decisiveness, I replied: "Moro".
Moro is a restaurant I knew mainly from its two cookbooks. Unlike many restaurant books I own which are geared more for the coffee table than the kitchen, both Moro cookbooks appear in my (albeit imagined) dinner plans often not only because they offer interpretations of the cuisines I love best – Spanish, North African and Middle Eastern – but also because the dishes they contain are simultaneously imaginative and unpretentious. I'm embarrassed to admit that I've managed only one recipe from the books (though I've admired others' efforts) but I knew that this was food I had to try at the source if ever given the chance. Still, I was well aware of the risk involved – I've learned to be wary of celebrity restaurants as fame has been known to dilute the very characteristics that made the restaurant great (not to mention inflate the prices), so it was with some trepidation that I called and wrangled us a table for that night.
As it turned out, my fears were completely ungrounded: the meal we had there qualified as one of the best I have ever eaten (not to mention offering astonishingly good value for money). The restaurant itself was warm and intimate but also relaxed enough to instantly dispel our concerns that we were a little underdressed (long-haul flight attire proving to not exactly translate into the best option for elegant dining). But most importantly, the food was exquisite. From my sauteed calves liver with cumin sauce, crispy lavash shards and cold yogurt to Manuel's meltingly tender pork belly with tangy green mojo verde, to the waitress-recommended Malaga raisin and sherry ice cream that had these self-confessed raisin-phobes fighting to lick the bowl, everything was a perfectly executed harmony of texture, temperature and flavor. There was also delicious fresh sheep's milk ricotta with baby fava beans and anchovies, wood-grilled Moroccan chicken with pine nuts, braised zucchini and caramelized-onion pilaf, and a silky and sour pistachio-yogurt-pomegranate cake. And there was bread, rough-hewn chunks of it, without a doubt among the finest specimens I have ever encountered: dense, brown, chewy, slightly sour and kissed with a breath of smoke from their wood-fired oven, it was bread that would have brought me to my knees had I been standing. We ate three baskets of it before our first courses even arrived, soaking it like a sponge in a dish of peppery Spanish olive oil. By the time we left, we were so full and happy that all we could murmur on our ride back to the hotel was how happy we were to trade a night in Jamaica for this.
At 7am the next morning, however, when no plane was at the gate, our optimism was starting to fade. And five hours later, it had all but disappeared. First there was the very late arrival of the inbound aircraft (a charter plane flown in from Atlanta on short notice), and then there was the problem of catering – I'm not sure what exactly the problem was, but it took several hours to resolve. We were hungry, tired and restless; we finished our light airport reading and had moved onto the heavy in-flight stuff. But much to our own surprise, we found we just couldn't get too despondent – we were, after all, surrounded by Jamaicans. Laughing and joking and facing this inconvenience with indefatigable optimism, they seduced us into sharing their simple outlook: if you can't change things, why get upset? And somehow we found ourselves not minding quite so much, even when our plane took off 23 hours later than originally scheduled, or even when they deposited us in Kingston instead of Montego Bay, or even when they made us all wait another five hours for the twenty-minute flight across the island.
But when we finally stumbled out into the humid night air at Montego Bay's Sangster International Airport, I can't tell you how happy I was to see a hand-lettered sign bearing my name. It was our Tourist Board-appointed driver, a smiling and gracious young man who introduced himself as Everton, packed us safely into his air-conditioned van, and whisked us away for the final leg of our journey.
the better part of two days. Despite the fact that he had been dutifully waiting for us all that time, however, he wouldn't let me even begin to apologize. Instead he shrugged, flashed us a wide smile and said, "Hey, no problem. I knew you would get here eventually."
It was a good omen – after all, the last thing we needed was more problems.
O-kaerinasai! (welcome back!) sounds like the startings of a great adventure! can't wait to hear more about it!
I have come to appreciate the traveling snafus that inevitably show up on vacation. Sometimes they don't translate well when told in a story, but you managed to incorporate the grace underlining the initial regret.The Red Stripe and the beach lounger are wonderful fodder for my imagination this afternoon.
Welcome back!Can´t wait to read more …..
great post…I see you adapted to the Jamacian way of life quite quickly! I'm also adding Moro to my restaurant list!
Where do I begin?!I can't believe the ordeal you endured just to get to Jamaica. But I must say you deserve full credit for dealing with it so positively.I have been a fan of the Moro cookbooks for the longest time, although I don't own them. But your description of your meal may mean that I'll have to add just a bit more to the load already threatening The Overburdened Bookshelf.I can't wait to read about the rest of your trip!
Jamaica, yeah, yeah, we're all jealous. But Moro, my dear? Oy vey, you have me crying in my soup over your good fortune.
I hope the rest of the trip goes (more) smoothly! Have fun. I'm looking forward to reading about all your adventures.
Sounds like your trip has been eventful to say the least. I am new to your blog and really enjoying reading it. Made your pesto the other day- yum! I am planning a trip to Europe next year- Moro is now well and truely on my list of restaurants to visit after your rave review. Hope the rest of your trip goes well after the rocky start.
Can't wait to read the rest of your story!!Welcome back!
Sorry to hear about the Heathrow delay – but sounds you had a fab holiday once you got to Jamaica. Looking forward to reading the stories and recipes – and drooling at the pictures taken by you and your hubby..


Hi everyone The off peak list of names has been added so you may contact each other if you may available. If anyone is not added and wishes to go on Any recent new members who wish to join this just mail text or leave message on the board. Best regards Andy bright Chairman 2016 community club of the year.