text:
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AP journalists posted at Ukraine's borders and within the country have put a human face to the mass movement of refugees, mostly women and children who have left their homes traumatized and exhausted, sometimes after being trapped for days or weeks in their basements to escape bombardment.
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Transforming Lactococcus lactis into a microbial cell factory
Kia Vest Petersen
Research output: Book/Report › Ph.D. thesis › Research
Biological conversion of lignocellulosic biomass to biofuels and -chemicals is a promising technology to reduce dependency on fossil fuels. This is important considering the environmental problems associated with consumption of the fossil fuels together with the fact that the reserves are limited and will be depleted if the increasing demand continues. However, one of the main challenges in the biological conversion is the identification of suitable platform organisms that can convert all the sugars present in the lignocellulosic biomass, including xylose. The aim of this PhD project was to investigate the potential of Lactococcus lactis as a platform organism for production of biofuels and-chemicals with a focus on characterization and optimization of the xylose metabolism. The plant isolate L. lactis KF147 was selected as the potential platform organism due to its natural ability to utilize both the pentose sugars xylose and arabinose. One of the desirable traits of a good platform organism is that it is easy to manipulate genetically. Since genetic manipulation usually involves introducing exogenous DNA, it is important that suitable methods are available. For this reason a standard protocol for preparing competent L. lactis KF147 cells was optimized resulting in a 100-fold increase in the transformation efficiency. Tools for introducing genes are likewise important. To expand the repertoire of genetic engineering tools available for L. lactis a novel tool named Repetitive Marker-Free Site-Specific Integration was developed. This tool facilitates repetitive rounds of site-specific integration of genes into the chromosome of L. lactis without leaving behind undesired vector elements. The site-specific integration is based on elements from the temperate lactococcal phage TP901-1, whereas excision of undesirable elements relies on a modified Cre-loxP system and 5-fluoroorotate mediated counter-selection. The plasmid used for the site-specific integration was termed pKV6 and when it is used for integrating genes, a new attachment site, attBmin, is likewise introduced, which can subsequently be used for the next round of integration. The xylose metabolism in L. lactis KF147 was characterized in a defined medium supplemented with 0.2%, 1% or 3% (w/v) xylose. The defined medium contains free arginine, and it was found that L. lactis KF147 co-metabolizes the arginine through the arginine deiminase pathway. To simplify further analysis arcA encoding the arginine deiminase was deleted, thus eliminating the arginine catabolism. We found that in L. lactis KF147 xylose is metabolized through two pathways namely the phosphoketolase pathway and the non-oxidative part of the pentose phosphate pathway. The only products formed were lactate, formate, acetate and ethanol, and the composition of the products depended on the xylose concentration. As xylose concentration increased, the proportion of xylose metabolized through the pentose phosphate pathway also increased. The effect from deleting and over-expressing the phosphoketolase pathway was also studied. The latter was achieved by introducing an additional copy of the ptk gene encoding the phosphoketolase. When the phosphoketolase pathway was deleted the product composition was similar to a typical mixed-acid pattern where formate, acetate and ethanol are formed in the ratio 2:1:1 in addition to lactate. For the strain with an inactive phosphoketolase pathway as well as for its parent, lactate production increased with xylose concentration, but in the phosphoketolase deficient strain up to almost three times more lactate was formed. In contrast when ptk was overexpressed the flux through the phosphoketolase pathway was 1.5 times higher compared to the parent strain when grown with 3% xylose. In addition, a clear shift towards a more mixed-acid fermentation profile was observed. The effect of adding additional arginine to the medium was likewise investigated in regards to growth of L. lactis KF147 and product composition. At low xylose concentration (0.2%) additional arginine greatly stimulated the growth of L. lactis KF147. In contrast, no effect of growth was observed at the higher xylose concentrations (1%, 3%). Irrespective of xylose concentration, arginine did affect the xylose and product fluxes, which all decreased as the arginine concentration increased. The final product yields were, however, not affected. L. lactis KF147 was also grown in rich M17 medium with xylose which showed that product composition strongly depends on the growth medium as the yield of lactate per xylose increase significantly. The pentose phosphate pathway present in L. lactis KF147 and other genome sequenced L. lactis strains is, however, a modified version of the known pentose phosphate pathway as no transaldolase gene is present in any of the these strains. A codon optimized version of the transaldolase gene ywjh from B. subtilis 168 was introduced in the phosphoketolase deficient strain. The effect of the introduced transaldolase gene was investigated in defined medium with 0.2% and 3% (w/v) xylose; however, no significant effects on either the growth rates or product formation were observed; even though expression of the introduced transaldolase was confirmed. The xylT gene, predicted to encode a D-xylose/H+-linked symporter, was deleted and the effect on growth evaluated in defined medium with 0.2% and 1% (w/v) xylose. Only at the low xylose concentration was a reduced growth rate observed for the ΔxylT strain compared to the parent strain. Based on this it was deduced that more than one transport system for xylose is present in L. lactis KF147. An adaptive evolution experiment was carried out with the goal of isolating mutants with improved growth on xylose. For this purpose the phosphoketolase deficient strain was applied. Of the 44 evolved strains screened for improved growth on xylose, three were selected for more detailed studies where specific growth rate and product formation were determined. These three strains underwent whole genome sequencing as well. The most interesting evolved strain was AD29 that, as the only of the investigated strains, exhibited both a pronounced accelerated growth on xylose (62.5% faster), and a changed fermentation profile with a clear increase in lactate production and corresponding drop in the production of formate, acetate, and ethanol. Three adaptive mutations were identified in AD29. Two is by all accounts involved in regulatory mechanisms either to stress (yhfB) or more globally (ytgF), and the last facilitate improved uptake of xylose (ptnC). Based on the above findings we conclude that L. lactis KF147 possesses many of the features a platform organism need, however whether the industry will find it attractive remains to be seen.
Department of Systems Biology, Technical University of Denmark
Fingerprint Dive into the research topics of 'Transforming Lactococcus lactis into a microbial cell factory'. Together they form a unique fingerprint.
Lactococcus lactis Agriculture & Biology
factories Agriculture & Biology
xylose Agriculture & Biology
phosphoketolase Agriculture & Biology
cells Agriculture & Biology
arginine Agriculture & Biology
transaldolase Agriculture & Biology
pentoses Agriculture & Biology
Petersen, K. V., Solem, C., Jensen, P. R., Martinussen, J., Mijakovic, I., Kok, J. & Jørgensen, S. T.
Petersen, K. V. (2014). Transforming Lactococcus lactis into a microbial cell factory. Department of Systems Biology, Technical University of Denmark.
Petersen, Kia Vest. / Transforming Lactococcus lactis into a microbial cell factory. Department of Systems Biology, Technical University of Denmark, 2014. 124 p.
@phdthesis{4388ae9aefaa4916a184e89e3cbdb877,
title = "Transforming Lactococcus lactis into a microbial cell factory",
abstract = "Biological conversion of lignocellulosic biomass to biofuels and -chemicals is a promising technology to reduce dependency on fossil fuels. This is important considering the environmental problems associated with consumption of the fossil fuels together with the fact that the reserves are limited and will be depleted if the increasing demand continues. However, one of the main challenges in the biological conversion is the identification of suitable platform organisms that can convert all the sugars present in the lignocellulosic biomass, including xylose. The aim of this PhD project was to investigate the potential of Lactococcus lactis as a platform organism for production of biofuels and-chemicals with a focus on characterization and optimization of the xylose metabolism. The plant isolate L. lactis KF147 was selected as the potential platform organism due to its natural ability to utilize both the pentose sugars xylose and arabinose. One of the desirable traits of a good platform organism is that it is easy to manipulate genetically. Since genetic manipulation usually involves introducing exogenous DNA, it is important that suitable methods are available. For this reason a standard protocol for preparing competent L. lactis KF147 cells was optimized resulting in a 100-fold increase in the transformation efficiency. Tools for introducing genes are likewise important. To expand the repertoire of genetic engineering tools available for L. lactis a novel tool named Repetitive Marker-Free Site-Specific Integration was developed. This tool facilitates repetitive rounds of site-specific integration of genes into the chromosome of L. lactis without leaving behind undesired vector elements. The site-specific integration is based on elements from the temperate lactococcal phage TP901-1, whereas excision of undesirable elements relies on a modified Cre-loxP system and 5-fluoroorotate mediated counter-selection. The plasmid used for the site-specific integration was termed pKV6 and when it is used for integrating genes, a new attachment site, attBmin, is likewise introduced, which can subsequently be used for the next round of integration. The xylose metabolism in L. lactis KF147 was characterized in a defined medium supplemented with 0.2%, 1% or 3% (w/v) xylose. The defined medium contains free arginine, and it was found that L. lactis KF147 co-metabolizes the arginine through the arginine deiminase pathway. To simplify further analysis arcA encoding the arginine deiminase was deleted, thus eliminating the arginine catabolism. We found that in L. lactis KF147 xylose is metabolized through two pathways namely the phosphoketolase pathway and the non-oxidative part of the pentose phosphate pathway. The only products formed were lactate, formate, acetate and ethanol, and the composition of the products depended on the xylose concentration. As xylose concentration increased, the proportion of xylose metabolized through the pentose phosphate pathway also increased. The effect from deleting and over-expressing the phosphoketolase pathway was also studied. The latter was achieved by introducing an additional copy of the ptk gene encoding the phosphoketolase. When the phosphoketolase pathway was deleted the product composition was similar to a typical mixed-acid pattern where formate, acetate and ethanol are formed in the ratio 2:1:1 in addition to lactate. For the strain with an inactive phosphoketolase pathway as well as for its parent, lactate production increased with xylose concentration, but in the phosphoketolase deficient strain up to almost three times more lactate was formed. In contrast when ptk was overexpressed the flux through the phosphoketolase pathway was 1.5 times higher compared to the parent strain when grown with 3% xylose. In addition, a clear shift towards a more mixed-acid fermentation profile was observed. The effect of adding additional arginine to the medium was likewise investigated in regards to growth of L. lactis KF147 and product composition. At low xylose concentration (0.2%) additional arginine greatly stimulated the growth of L. lactis KF147. In contrast, no effect of growth was observed at the higher xylose concentrations (1%, 3%). Irrespective of xylose concentration, arginine did affect the xylose and product fluxes, which all decreased as the arginine concentration increased. The final product yields were, however, not affected. L. lactis KF147 was also grown in rich M17 medium with xylose which showed that product composition strongly depends on the growth medium as the yield of lactate per xylose increase significantly. The pentose phosphate pathway present in L. lactis KF147 and other genome sequenced L. lactis strains is, however, a modified version of the known pentose phosphate pathway as no transaldolase gene is present in any of the these strains. A codon optimized version of the transaldolase gene ywjh from B. subtilis 168 was introduced in the phosphoketolase deficient strain. The effect of the introduced transaldolase gene was investigated in defined medium with 0.2% and 3% (w/v) xylose; however, no significant effects on either the growth rates or product formation were observed; even though expression of the introduced transaldolase was confirmed. The xylT gene, predicted to encode a D-xylose/H+-linked symporter, was deleted and the effect on growth evaluated in defined medium with 0.2% and 1% (w/v) xylose. Only at the low xylose concentration was a reduced growth rate observed for the ΔxylT strain compared to the parent strain. Based on this it was deduced that more than one transport system for xylose is present in L. lactis KF147. An adaptive evolution experiment was carried out with the goal of isolating mutants with improved growth on xylose. For this purpose the phosphoketolase deficient strain was applied. Of the 44 evolved strains screened for improved growth on xylose, three were selected for more detailed studies where specific growth rate and product formation were determined. These three strains underwent whole genome sequencing as well. The most interesting evolved strain was AD29 that, as the only of the investigated strains, exhibited both a pronounced accelerated growth on xylose (62.5% faster), and a changed fermentation profile with a clear increase in lactate production and corresponding drop in the production of formate, acetate, and ethanol. Three adaptive mutations were identified in AD29. Two is by all accounts involved in regulatory mechanisms either to stress (yhfB) or more globally (ytgF), and the last facilitate improved uptake of xylose (ptnC). Based on the above findings we conclude that L. lactis KF147 possesses many of the features a platform organism need, however whether the industry will find it attractive remains to be seen.",
author = "Petersen, {Kia Vest}",
publisher = "Department of Systems Biology, Technical University of Denmark",
Petersen, KV 2014, Transforming Lactococcus lactis into a microbial cell factory. Department of Systems Biology, Technical University of Denmark.
Transforming Lactococcus lactis into a microbial cell factory. / Petersen, Kia Vest.
Department of Systems Biology, Technical University of Denmark, 2014. 124 p.
T1 - Transforming Lactococcus lactis into a microbial cell factory
AU - Petersen, Kia Vest
N2 - Biological conversion of lignocellulosic biomass to biofuels and -chemicals is a promising technology to reduce dependency on fossil fuels. This is important considering the environmental problems associated with consumption of the fossil fuels together with the fact that the reserves are limited and will be depleted if the increasing demand continues. However, one of the main challenges in the biological conversion is the identification of suitable platform organisms that can convert all the sugars present in the lignocellulosic biomass, including xylose. The aim of this PhD project was to investigate the potential of Lactococcus lactis as a platform organism for production of biofuels and-chemicals with a focus on characterization and optimization of the xylose metabolism. The plant isolate L. lactis KF147 was selected as the potential platform organism due to its natural ability to utilize both the pentose sugars xylose and arabinose. One of the desirable traits of a good platform organism is that it is easy to manipulate genetically. Since genetic manipulation usually involves introducing exogenous DNA, it is important that suitable methods are available. For this reason a standard protocol for preparing competent L. lactis KF147 cells was optimized resulting in a 100-fold increase in the transformation efficiency. Tools for introducing genes are likewise important. To expand the repertoire of genetic engineering tools available for L. lactis a novel tool named Repetitive Marker-Free Site-Specific Integration was developed. This tool facilitates repetitive rounds of site-specific integration of genes into the chromosome of L. lactis without leaving behind undesired vector elements. The site-specific integration is based on elements from the temperate lactococcal phage TP901-1, whereas excision of undesirable elements relies on a modified Cre-loxP system and 5-fluoroorotate mediated counter-selection. The plasmid used for the site-specific integration was termed pKV6 and when it is used for integrating genes, a new attachment site, attBmin, is likewise introduced, which can subsequently be used for the next round of integration. The xylose metabolism in L. lactis KF147 was characterized in a defined medium supplemented with 0.2%, 1% or 3% (w/v) xylose. The defined medium contains free arginine, and it was found that L. lactis KF147 co-metabolizes the arginine through the arginine deiminase pathway. To simplify further analysis arcA encoding the arginine deiminase was deleted, thus eliminating the arginine catabolism. We found that in L. lactis KF147 xylose is metabolized through two pathways namely the phosphoketolase pathway and the non-oxidative part of the pentose phosphate pathway. The only products formed were lactate, formate, acetate and ethanol, and the composition of the products depended on the xylose concentration. As xylose concentration increased, the proportion of xylose metabolized through the pentose phosphate pathway also increased. The effect from deleting and over-expressing the phosphoketolase pathway was also studied. The latter was achieved by introducing an additional copy of the ptk gene encoding the phosphoketolase. When the phosphoketolase pathway was deleted the product composition was similar to a typical mixed-acid pattern where formate, acetate and ethanol are formed in the ratio 2:1:1 in addition to lactate. For the strain with an inactive phosphoketolase pathway as well as for its parent, lactate production increased with xylose concentration, but in the phosphoketolase deficient strain up to almost three times more lactate was formed. In contrast when ptk was overexpressed the flux through the phosphoketolase pathway was 1.5 times higher compared to the parent strain when grown with 3% xylose. In addition, a clear shift towards a more mixed-acid fermentation profile was observed. The effect of adding additional arginine to the medium was likewise investigated in regards to growth of L. lactis KF147 and product composition. At low xylose concentration (0.2%) additional arginine greatly stimulated the growth of L. lactis KF147. In contrast, no effect of growth was observed at the higher xylose concentrations (1%, 3%). Irrespective of xylose concentration, arginine did affect the xylose and product fluxes, which all decreased as the arginine concentration increased. The final product yields were, however, not affected. L. lactis KF147 was also grown in rich M17 medium with xylose which showed that product composition strongly depends on the growth medium as the yield of lactate per xylose increase significantly. The pentose phosphate pathway present in L. lactis KF147 and other genome sequenced L. lactis strains is, however, a modified version of the known pentose phosphate pathway as no transaldolase gene is present in any of the these strains. A codon optimized version of the transaldolase gene ywjh from B. subtilis 168 was introduced in the phosphoketolase deficient strain. The effect of the introduced transaldolase gene was investigated in defined medium with 0.2% and 3% (w/v) xylose; however, no significant effects on either the growth rates or product formation were observed; even though expression of the introduced transaldolase was confirmed. The xylT gene, predicted to encode a D-xylose/H+-linked symporter, was deleted and the effect on growth evaluated in defined medium with 0.2% and 1% (w/v) xylose. Only at the low xylose concentration was a reduced growth rate observed for the ΔxylT strain compared to the parent strain. Based on this it was deduced that more than one transport system for xylose is present in L. lactis KF147. An adaptive evolution experiment was carried out with the goal of isolating mutants with improved growth on xylose. For this purpose the phosphoketolase deficient strain was applied. Of the 44 evolved strains screened for improved growth on xylose, three were selected for more detailed studies where specific growth rate and product formation were determined. These three strains underwent whole genome sequencing as well. The most interesting evolved strain was AD29 that, as the only of the investigated strains, exhibited both a pronounced accelerated growth on xylose (62.5% faster), and a changed fermentation profile with a clear increase in lactate production and corresponding drop in the production of formate, acetate, and ethanol. Three adaptive mutations were identified in AD29. Two is by all accounts involved in regulatory mechanisms either to stress (yhfB) or more globally (ytgF), and the last facilitate improved uptake of xylose (ptnC). Based on the above findings we conclude that L. lactis KF147 possesses many of the features a platform organism need, however whether the industry will find it attractive remains to be seen.
AB - Biological conversion of lignocellulosic biomass to biofuels and -chemicals is a promising technology to reduce dependency on fossil fuels. This is important considering the environmental problems associated with consumption of the fossil fuels together with the fact that the reserves are limited and will be depleted if the increasing demand continues. However, one of the main challenges in the biological conversion is the identification of suitable platform organisms that can convert all the sugars present in the lignocellulosic biomass, including xylose. The aim of this PhD project was to investigate the potential of Lactococcus lactis as a platform organism for production of biofuels and-chemicals with a focus on characterization and optimization of the xylose metabolism. The plant isolate L. lactis KF147 was selected as the potential platform organism due to its natural ability to utilize both the pentose sugars xylose and arabinose. One of the desirable traits of a good platform organism is that it is easy to manipulate genetically. Since genetic manipulation usually involves introducing exogenous DNA, it is important that suitable methods are available. For this reason a standard protocol for preparing competent L. lactis KF147 cells was optimized resulting in a 100-fold increase in the transformation efficiency. Tools for introducing genes are likewise important. To expand the repertoire of genetic engineering tools available for L. lactis a novel tool named Repetitive Marker-Free Site-Specific Integration was developed. This tool facilitates repetitive rounds of site-specific integration of genes into the chromosome of L. lactis without leaving behind undesired vector elements. The site-specific integration is based on elements from the temperate lactococcal phage TP901-1, whereas excision of undesirable elements relies on a modified Cre-loxP system and 5-fluoroorotate mediated counter-selection. The plasmid used for the site-specific integration was termed pKV6 and when it is used for integrating genes, a new attachment site, attBmin, is likewise introduced, which can subsequently be used for the next round of integration. The xylose metabolism in L. lactis KF147 was characterized in a defined medium supplemented with 0.2%, 1% or 3% (w/v) xylose. The defined medium contains free arginine, and it was found that L. lactis KF147 co-metabolizes the arginine through the arginine deiminase pathway. To simplify further analysis arcA encoding the arginine deiminase was deleted, thus eliminating the arginine catabolism. We found that in L. lactis KF147 xylose is metabolized through two pathways namely the phosphoketolase pathway and the non-oxidative part of the pentose phosphate pathway. The only products formed were lactate, formate, acetate and ethanol, and the composition of the products depended on the xylose concentration. As xylose concentration increased, the proportion of xylose metabolized through the pentose phosphate pathway also increased. The effect from deleting and over-expressing the phosphoketolase pathway was also studied. The latter was achieved by introducing an additional copy of the ptk gene encoding the phosphoketolase. When the phosphoketolase pathway was deleted the product composition was similar to a typical mixed-acid pattern where formate, acetate and ethanol are formed in the ratio 2:1:1 in addition to lactate. For the strain with an inactive phosphoketolase pathway as well as for its parent, lactate production increased with xylose concentration, but in the phosphoketolase deficient strain up to almost three times more lactate was formed. In contrast when ptk was overexpressed the flux through the phosphoketolase pathway was 1.5 times higher compared to the parent strain when grown with 3% xylose. In addition, a clear shift towards a more mixed-acid fermentation profile was observed. The effect of adding additional arginine to the medium was likewise investigated in regards to growth of L. lactis KF147 and product composition. At low xylose concentration (0.2%) additional arginine greatly stimulated the growth of L. lactis KF147. In contrast, no effect of growth was observed at the higher xylose concentrations (1%, 3%). Irrespective of xylose concentration, arginine did affect the xylose and product fluxes, which all decreased as the arginine concentration increased. The final product yields were, however, not affected. L. lactis KF147 was also grown in rich M17 medium with xylose which showed that product composition strongly depends on the growth medium as the yield of lactate per xylose increase significantly. The pentose phosphate pathway present in L. lactis KF147 and other genome sequenced L. lactis strains is, however, a modified version of the known pentose phosphate pathway as no transaldolase gene is present in any of the these strains. A codon optimized version of the transaldolase gene ywjh from B. subtilis 168 was introduced in the phosphoketolase deficient strain. The effect of the introduced transaldolase gene was investigated in defined medium with 0.2% and 3% (w/v) xylose; however, no significant effects on either the growth rates or product formation were observed; even though expression of the introduced transaldolase was confirmed. The xylT gene, predicted to encode a D-xylose/H+-linked symporter, was deleted and the effect on growth evaluated in defined medium with 0.2% and 1% (w/v) xylose. Only at the low xylose concentration was a reduced growth rate observed for the ΔxylT strain compared to the parent strain. Based on this it was deduced that more than one transport system for xylose is present in L. lactis KF147. An adaptive evolution experiment was carried out with the goal of isolating mutants with improved growth on xylose. For this purpose the phosphoketolase deficient strain was applied. Of the 44 evolved strains screened for improved growth on xylose, three were selected for more detailed studies where specific growth rate and product formation were determined. These three strains underwent whole genome sequencing as well. The most interesting evolved strain was AD29 that, as the only of the investigated strains, exhibited both a pronounced accelerated growth on xylose (62.5% faster), and a changed fermentation profile with a clear increase in lactate production and corresponding drop in the production of formate, acetate, and ethanol. Three adaptive mutations were identified in AD29. Two is by all accounts involved in regulatory mechanisms either to stress (yhfB) or more globally (ytgF), and the last facilitate improved uptake of xylose (ptnC). Based on the above findings we conclude that L. lactis KF147 possesses many of the features a platform organism need, however whether the industry will find it attractive remains to be seen.
M3 - Ph.D. thesis
BT - Transforming Lactococcus lactis into a microbial cell factory
PB - Department of Systems Biology, Technical University of Denmark
Petersen KV. Transforming Lactococcus lactis into a microbial cell factory. Department of Systems Biology, Technical University of Denmark, 2014. 124 p.

redpajama_set_name: RedPajamaCommonCrawl


Goldratt's Theory of Constraints (TOC) is usually thought of when considering equipment or labor as the constraining factor limiting output. However, the supply chain can also act as the constraint. You might have a single supplier whose capacity to deliver what you need is limited. It is important to quickly recognize this situation and use the drum–buffer–rope technique to optimize your supply chain and your ERP to optimize the process.
If your supplier is your constraint, then every action and decision will be subservient to that constraint. Use your manufacturing ERP to identify the maximum rate of production they are capable of. That rate is your drumbeat. Your own internal processes may go faster. Other suppliers may go faster. It doesn't make any difference; whatever rate that supplier is capable of, that is your limit and making other operations run faster only builds inventory.
Once the drum rate is understood, begin to establish buffers ahead of your constraint. A buffer could be inventory or help with raw materials so your supplier never runs out of any necessary inventory and can keep producing. The buffer could be working capital to help ensure production never falls below the drum rate.
Transportation is another common supply chain constraint. We have all seen images of the giant crawler vehicles that moved the space shuttle to its launch platform. The movement is in meters, not miles, per hour. A buffer here might be space. Anything that brings the starting and finishing points closer together will help your cause.
The final element in the TOC process is the rope. Link your processes together so that none of them move faster than the constraint on the upstream side. Think of the rope as a limit on other processes and suppliers that ties them to the constraint's drum rate. Avoid bringing in component materials from other suppliers ahead of the constraint, since they cannot be used yet. Similarly, starting work on another subassembly in house that will not be used until the constrained material is available will add no value and may cost you.
Thoughtful use of ERP will coordinate supply chain components with your constraints wherever you find them.
Your manufacturing ERP should contain the expected lead times for all material parts. The requested date and quantity should also be recorded in your ERP system. As purchase orders progress, those requested dates and quantities will be supplanted by promised dates and quantities and the ASN should be the final data element for planning. Thoughtful use of ERP will coordinate supply chain components with your constraints wherever you find them. You already have the tools you need, now start using them to optimize your constrained supply chain the TOC way.


On-site wastewater treatment is an option if there is no sewer available or if the owner wants to recycle water to reduce demand on mains wastewater systems.
The Building Code requires that if a sewer is available the drainage system must be connected to it – although the local authority can provide a waiver, allowing building owners to reduce demand on mains systems by recycling greywater or using a composting toilet.
If there is no sewer available, on-site treatment options will be needed.
With all wastewater disposal or recycling, health and safety must be the overriding priority.
Installing an on-site wastewater treatment system requires a building consent and the installation must be carried out or supervised by a certified drainlayer.
Some local authorities have specific requirements that homeowners must comply with. For example, Auckland Council requires that all wastewater treatment and disposal systems in its area are serviced at least every six months.
Blackwater is wastewater from toilets, dishwashing machines and sinks – because the fats, detergents and cleaning agents used in kitchen wastewater, this is considered blackwater and must be discharged accordingly.
Greywater is waste from baths, showers and hand basins.
Wastewater from clothes washing machines may be considered in either category.
An on-site wastewater treatment system must meet performance requirements for construction and operation of the Building Code. If the system is designed to AS/NZS 1547:2012 On-site domestic-wastewater management, the requirements of the Code will be met.
As noted above, the Building Code requires that if a sewer is available the drainage system must be connected to it – but the local authority can provide a waiver allowing on-site wastewater treatment to be installed. Local authorities or regional councils may have additional bylaws controlling the installation of on-site wastewater treatment systems. The local authority should be contacted before you begin to design a system.
Septic tank construction is covered by AS/NZS 1546.1:2008 On-site domestic wastewater treatment units – Septic Tanks.
Most on-site wastewater treatment systems involve two stages of treatment – the first stage in a tank or treatment system, and the second when the effluent is dispersed on to land or the garden and further breaks down. The first stage may be carried out in a septic tank or in a more advanced system such as an aerated wastewater treatment system or advanced sewage treatment system. These systems are much more advanced than septic tanks and treat effluent to a level that allows it to be used on the garden or even recycled for toilet flushing and vehicle washing.
For new building projects, most local and regional councils require a higher level of wastewater treatment than that provided by the traditional septic tank.
Both of these options reduce the amount of effluent to be disposed of by the wastewater treatment system.
Investigate the power consumption and maintenance costs before specifying a model of sewage disposal system – these can vary greatly and can have a big impact on the ongoing costs for the owner.
front-loading washing machine rather than top-loading machine.
Reusing greywater in garden irrigation and, if treated, for toilet flushing, is another good way to reduce the volume of liquid going to an on-site wastewater treatment system.


BLEU (bilingual evaluation understudy) is an algorithm for evaluating the quality of text which has been machine-translated from one natural language to another. Quality is considered to be the correspondence between a machine's output and that of a human: "the closer a machine translation is to a professional human translation, the better it is" – this is the central idea behind BLEU. BLEU was one of the first metrics to achieve a high correlation with human judgements of quality, and remains one of the most popular automated and inexpensive metrics.
Scores are calculated for individual translated segments—generally sentences—by comparing them with a set of good quality reference translations. Those scores are then averaged over the whole corpus to reach an estimate of the translation's overall quality. Intelligibility or grammatical correctness are not taken into account.
BLEU is designed to approximate human judgement at a corpus level, and performs badly if used to evaluate the quality of individual sentences.
BLEU's output is always a number between 0 and 1. This value indicates how similar the candidate and reference texts are, with values closer to 1 representing more similar texts. However, few human translations will attain a score of 1. The candidate texts must be identical to a reference translation. For this reason, it is not necessary to attain a score of 1. Because there are more opportunities to match, adding additional reference translations will increase the BLEU score.
where is number of words from the candidate that are found in the reference, and is the total number of words in the candidate. This is a perfect score, despite the fact that the candidate translation above retains little of the content of either of the references.
The modification that BLEU makes is fairly straightforward. For each word in the candidate translation, the algorithm takes its maximum total count, , in any of the reference translations. In the example above, the word "the" appears twice in reference 1, and once in reference 2. Thus .
In practice, however, using individual words as the unit of comparison is not optimal. Instead, BLEU computes the same modified precision metric using n-grams. The length which has the "highest correlation with monolingual human judgements" was found to be four. The unigram scores are found to account for the adequacy of the translation, how much information is retained. The longer -gram scores account for the fluency of the translation, or to what extent it reads like "good English".
as the word 'the' and the word 'cat' appear once each in the candidate, and the total number of words is two. The modified bigram precision would be as the bigram, "the cat" appears once in the candidate. It has been pointed out that precision is usually twinned with recall to overcome this problem, as the unigram recall of this example would be or . The problem being that as there are multiple reference translations, a bad translation could easily have an inflated recall, such as a translation which consisted of all the words in each of the references.
iBLEU is an interactive version of BLEU that allows a user to visually examine the BLEU scores obtained by the candidate translations. It also allows comparing two different systems in a visual and interactive manner which is useful for system development.
It has been argued that although BLEU has significant advantages, there is no guarantee that an increase in BLEU score is an indicator of improved translation quality. There is an inherent, systemic problem with any metric based on comparing with one or a few reference translations: in real life, sentences can be translated in many different ways, sometimes with no overlap. Therefore, the approach of comparing by how much any given translation result by a computer differs from just a few human translations is flawed. HyTER is another automated MT metric that compares to very many translations in a reference grammar defined by human translators; the drawback is then that the human effort involved in correctly defining the combinatorially many ways to render the meaning of the translation in practice means HyTER also is only an approximation.


Three years ago I met and fell in love with the most wonderful man. In fact, I would even say he is the man of my dreams. Two years into our courtship, he asked me to marry him and I gladly accepted. This June we will celebrate our one-year anniversary. Our life is good. We both have great careers and soon we will move into our dream home.
While dating, my husband and I both shared the position that we did not want kids. Moreover, we agreed that if either one of us changed our position, adoption would be our first option. Well, during the holiday season of 2012, my husband's father passed away and through his grieving process, my husband decided that a great way for him to honor the memory of his father is to have a child naturally - perhaps even a boy he could name after his father. There's only one problem: I am Transgender. Which means, in my case, I was born male and underwent the necessary medical procedures to become female.
I appreciate you for being so open and allowing your heart to shine through. Obviously, you are deeply in love with your husband and I can see your pain is wearing on your heart. Shame can wear on you like a winter coat in the hot summer sun and have you sweating coffee and tea, girl.
Honesty is always the best policy. However, in this case I have to ask myself if it will cause more harm than help. With that being said, reality now has to set in. You have two issues that you have to face and deal with. You deceived your husband, and now with the passing of his father his heart is truly on the line. You have to ask yourself, 'If I tell him, what would it do to him?' Everyone knows about the two of you - his family, friends, co-workers. Not saying that there is a charade but if you are a 'real' woman it's time to act like it!
The past is behind you and you cannot change that. Now, is telling him worth destroying him just because you need to get it off your chest? It's time to put your big-girl drawers on and take responsibly for the present situation. Forgive me for being a little harsh but you have to realize that it's not about you. The situation may deal with you, but this is about him and where he now is with his life with you. He loves you so much he wants to have a child with you. Once again, asking for two to become one. Having a child together bonds you in ways that can only be explained by the experience.
When you are in a relationship, you are entrusted with another's life. Remember those wedding vows. You were not alone on that day, and you have to take that to heart. You have to come outside of yourself and see his life, not just yours. Remember the love you have for him and that you never want to hurt him.
Stick to the facts, and display all of them to him. Fact #1: You cannot have children! Fact #2: You were ashamed to tell him that you could not mother a child. For that is the reason adoption was the option if, in fact, you ever wanted children in your lives. Another option is surrogacy, which would allow you both to take part in the process. If you tell him your 'little secret,' his life will forever be changed. Are you willing to take that risk?
Love is a very wondrous thing that makes you look past yourself to see the greater good. You look past your pain to see his. You speak so highly of him and the love you have for him is regal. Cherish the love you have and be honest with him from here on out. Remember, the way it begins is the way it will end.
I so love you for being comfortable in your own skin! The fact that you come outside of yourself to truly appreciate and embrace the racy, radical culture of female Illusion does my heart good. Being an old showgirl myself I surely can appreciate you. Girl, you have made my day! This is the best thing ever! Female Illusionists/Drag Queens want to look like women (all variations). Glamazon, fantasy, vixen, or the girl next door, Queens/Illusionists have developed an art to look like women. To have a biological woman who has an obsession with the Queen culture and truly embraced the culture is wonderful to hear.
Now, to answer your question, yes, I think spiritually you are Trans. You have officially joined the 'Trans Band.' So, girl, talk your favorite Queen/Queer, get the latest fashion, get your hair/nails done, head out and enjoy all that Seattle has to offer, and twerk it! Now we all love fun and games but let us not forget the 'real' thought. I hope you are supporting the community - we can always use as much support as possible. Pride is coming up and I know you don't want to miss it. Check with SGN for all the latest and updated news about Pride events. You have the opportunity to get all dolled up in whatever your heart desires and come out to show your support and show some love for the LGBT community.


text:
package com.google.code.play2.plugin;

import java.io.File;
import java.io.IOException;
import java.util.Arrays;

import org.apache.maven.plugin.MojoExecutionException;
import org.apache.maven.plugins.annotations.Component;
import org.apache.maven.plugins.annotations.LifecyclePhase;
import org.apache.maven.plugins.annotations.Mojo;
import org.apache.maven.plugins.annotations.Parameter;
import org.sonatype.plexus.build.incremental.BuildContext;

import com.google.code.play2.provider.api.AssetCompilationException;
import com.google.code.play2.provider.api.JavascriptCompilationResult;
import com.google.code.play2.provider.api.Play2JavascriptCompiler;
import com.google.code.play2.provider.api.Play2Provider;

/**
 * Compile JavaScript assets
 *
 * @author Grzegorz Slowikowski
 * @since 1.0.0
 */
@Mojo( name = "closure-compile", defaultPhase = LifecyclePhase.GENERATE_RESOURCES )
public class Play2ClosureCompileMojo
    extends AbstractPlay2AssetsCompileMojo
{
    /**
     * Javascript compiler entry points includes, separated by commas.
     *
     * @since 1.0.0
     */
    @Parameter( property = "play2.javascriptEntryPointsIncludes", defaultValue = "**/*.js" )
    private String javascriptEntryPointsIncludes;

    /**
     * Javascript compiler entry points excludes, separated by commas.
     *
     * @since 1.0.0
     */
    @Parameter( property = "play2.javascriptEntryPointsExcludes", defaultValue = "**/_*" )
    private String javascriptEntryPointsExcludes;

    /**
     * Javascript compiler options, separated by spaces.
     *
     * @since 1.0.0
     */
    @Parameter( property = "play2.closureCompilerOptions", defaultValue = "" )
    private String closureCompilerOptions;

    /**
     * For M2E integration.
     */
    @Component
    private BuildContext buildContext;

    @Override
    protected String getAssetsIncludes()
    {
        return javascriptEntryPointsIncludes;
    }

    @Override
    protected String getAssetsExcludes()
    {
        return javascriptEntryPointsExcludes;
    }

    @Override
    protected void compileAssets( File assetsSourceDirectory, String[] fileNames, File outputDirectory )
        throws AssetCompilationException, IOException, MojoExecutionException
    {
        int compiledFiles = 0;

        Play2Provider play2Provider = getProvider();
        Play2JavascriptCompiler compiler = play2Provider.getJavascriptCompiler();
        if ( closureCompilerOptions != null )
        {
            compiler.setCompilerOptions( Arrays.asList( closureCompilerOptions.split( " " ) ) );
        }

        for ( String fileName : fileNames )
        {
            File srcJsFile = new File( assetsSourceDirectory, fileName );

            // String jsFileName = fileName.replace( ".coffee", ".js" );
            File jsFile = new File( outputDirectory, fileName/* jsFileName */ );

            String minifiedJsFileName = fileName.replace( ".js", ".min.js" );
            File minifiedJsFile = new File( outputDirectory, minifiedJsFileName );

            boolean modified = true;
            if ( jsFile.isFile() )
            {
                modified = jsFile.lastModified() < srcJsFile.lastModified();
            }

            if ( modified )
            {
                getLog().debug( String.format( "Processing \"%s\"", fileName ) );

                JavascriptCompilationResult result = compiler.compile( srcJsFile );
                String jsContent = result.getJs();
                String minifiedJsContent = result.getMinifiedJs();
                createDirectory( jsFile.getParentFile(), false );
                writeToFile( jsFile, "UTF-8", jsContent );
                buildContext.refresh( jsFile );

                if ( minifiedJsContent != null )
                {
                    createDirectory( minifiedJsFile.getParentFile(), false );
                    writeToFile( minifiedJsFile, "UTF-8", minifiedJsContent );
                    buildContext.refresh( minifiedJsFile );
                }
                else
                {
                    if ( minifiedJsFile.exists() && minifiedJsFile.isFile() && minifiedJsFile.delete() )
                    {
                        buildContext.refresh( minifiedJsFile );
                    }
                }
                compiledFiles++;
            }
            else
            {
                getLog().debug( String.format( "\"%s\" skipped - no changes", fileName ) );
            }
        }

        getLog().info( String.format( "%d %s processed, %d compiled", Integer.valueOf( fileNames.length ),
                                      fileNames.length > 1 ? "assets" : "asset", Integer.valueOf( compiledFiles ) ) );
    }

}