Difference between revisions of "Team:Aalto-Helsinki/Project"
m (marginals of one pic) |
m (E. coli in italics) |
||
(10 intermediate revisions by 4 users not shown) | |||
Line 79: | Line 79: | ||
<p>While power generation accounts for about a quarter of the world’s greenhouse gas emissions, 11% might seem like a small number. What makes emissions made by transportation significant, though, is that there are currently no good alternatives for gasoline. Electric cars are emerging, but they still have quite a way to go before reaching the price-range suitable for middle class. Propane is already widely used as a <a href="http://www.iea.org/publications/globalevoutlook_2013.pdf" target="_blank">replacement of gasoline</a>. In South Korea, <a href="http://www.auto-gas.net/uploads/Autogas%20Incentive%20Policies%202014.pdf" target="_blank">2.2 million</a> vehicles run on propane and in Turkey, 37% of passenger cars use it. On a large scale though, <a href="http://www.auto-gas.net/uploads/Autogas%20Incentive%20Policies%202014.pdf" target="_blank">only 1.2%</a> of vehicles worldwide are suitable to run on propane. This may seem discouraging, but in reality converting a gasoline motor into a propane one is quite simple and inexpensive. DIY converter kits are sold online for less than $500. In Canada, depending on the vehicle type, conversion done by a third party costs somewhere around <a href="http://www.autopropane.com/html/faqs.html" target="_blank">2500-6500 dollars</a>.</p> | <p>While power generation accounts for about a quarter of the world’s greenhouse gas emissions, 11% might seem like a small number. What makes emissions made by transportation significant, though, is that there are currently no good alternatives for gasoline. Electric cars are emerging, but they still have quite a way to go before reaching the price-range suitable for middle class. Propane is already widely used as a <a href="http://www.iea.org/publications/globalevoutlook_2013.pdf" target="_blank">replacement of gasoline</a>. In South Korea, <a href="http://www.auto-gas.net/uploads/Autogas%20Incentive%20Policies%202014.pdf" target="_blank">2.2 million</a> vehicles run on propane and in Turkey, 37% of passenger cars use it. On a large scale though, <a href="http://www.auto-gas.net/uploads/Autogas%20Incentive%20Policies%202014.pdf" target="_blank">only 1.2%</a> of vehicles worldwide are suitable to run on propane. This may seem discouraging, but in reality converting a gasoline motor into a propane one is quite simple and inexpensive. DIY converter kits are sold online for less than $500. In Canada, depending on the vehicle type, conversion done by a third party costs somewhere around <a href="http://www.autopropane.com/html/faqs.html" target="_blank">2500-6500 dollars</a>.</p> | ||
− | <p>On top of its use as a transportation fuel, propane is also a popular cooking fuel in developing countries. It’s more commonly used in urban areas, as access to propane can be difficult in rural areas. Nevertheless, according to the International Energy Association (IEA), about 20% of household in the rural areas of Botswana use propane as their source of energy. In the urban areas the usage is around 60%. | + | <p>On top of its use as a transportation fuel, propane is also a popular cooking fuel in developing countries. It’s more commonly used in urban areas, as access to propane can be difficult in rural areas. Nevertheless, according to the International Energy Association (IEA), about 20% of household in the rural areas of Botswana use propane as their source of energy. In the urban areas the usage is around 60%. Expensive infrastructure is in place for example in Brazil, where <a href="https://www.iea.org/publications/freepublications/publication/cooking.pdf" target="_blank" style="padding:0">98% of households</a> have access to propane due to government funded efforts. One major goal of promoting propane is to replace currently used biomass fuels, including wood. Wood consumption is often unsustainable and threatens the local ecosystems. Additionally, the traditional fuels (biomass and coal) produce high emissions of carbon monoxide, hydrocarbons and particulate matter. IEA suggests that these impurities are responsible for <a href="https://www.iea.org/publications/freepublications/publication/cooking.pdf" target="_blank" style="padding:0">more premature deaths</a> than malaria in developing countries.</p> |
− | <p>Propane can be considered <a href="http://pubs.acs.org/doi/abs/10.1021/ef5006379" target="_blank">a clean fuel </a>, as it emits less CO\(_2\) than gasoline or ethanol and, when compared with traditional biomasses, has significantly smaller emission factors based on both mass and energy delivery. Thus, propane is a viable option for replacing traditional biomasses as cooking fuel, although currently the price is not competitive without government subsidies.</p> | + | <p>Propane can be considered <a href="http://pubs.acs.org/doi/abs/10.1021/ef5006379" target="_blank">a clean fuel</a>, as it emits less CO\(_2\) than gasoline or ethanol and, when compared with traditional biomasses, has significantly smaller emission factors based on both mass and energy delivery. Thus, propane is a viable option for replacing traditional biomasses as cooking fuel, although currently the price is not competitive without government subsidies.</p> |
<div class="row"> | <div class="row"> | ||
<figure style="margin: 10px;"> | <figure style="margin: 10px;"> | ||
<a href="https://static.igem.org/mediawiki/2015/c/c6/Aalto-Helsinki_emissioncharts.png" target="_blank"><img src="https://static.igem.org/mediawiki/2015/c/c6/Aalto-Helsinki_emissioncharts.png" alt="Emission charts" style="max-height: 100%; max-width: 100%;"/></a> | <a href="https://static.igem.org/mediawiki/2015/c/c6/Aalto-Helsinki_emissioncharts.png" target="_blank"><img src="https://static.igem.org/mediawiki/2015/c/c6/Aalto-Helsinki_emissioncharts.png" alt="Emission charts" style="max-height: 100%; max-width: 100%;"/></a> | ||
− | <figcaption style="font-size:12px;"><b>Figure 1.</b> | + | <figcaption style="font-size:12px;"><b>Figure 1.</b> <a href="http://pubs.acs.org/doi/abs/10.1021/ef5006379" target="_blank">Comparison of various fuels</a> by delivered-energy-based CO and particle matter emissions (EF, g/MJ). The y axis is shown in the log scale.</figcaption> |
</figure> | </figure> | ||
</div> | </div> | ||
− | <p>However, the propane we are currently using is produced as a side product of the petrochemical industry. This | + | <p>However, the propane we are currently using is produced as a side product of the petrochemical industry. This essentially means that propane is a fossil fuel, emitting stored CO\(_2\) into the atmosphere without a way of returning to the natural carbon cycle. This contributes not only to climate change but also ocean acidification. This endangers <a href="http://www.sciencedirect.com/science/article/pii/S1877343512000620" target="_blank">the livelihoods </a>of hundreds of millions of people directly or indirectly dependent on the marine ecosystems. </p> |
<p>We want to make the use of propane as a fuel sustainable. We want to design an <i>Eschericia coli</i> capable of producing propane from cellulose. </p> | <p>We want to make the use of propane as a fuel sustainable. We want to design an <i>Eschericia coli</i> capable of producing propane from cellulose. </p> | ||
Line 109: | Line 109: | ||
</figure> | </figure> | ||
− | <p>Our propane pathway is based on the research done by <a href="http://www.nature.com/ncomms/2014/140902/ncomms5731/full/ncomms5731.html" target="_blank">Dr. Pauli Kallio <i>et al.</i></a> and <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61" target="_blank">Menon <i>et al.</i></a>. Right after we decided our topic we got in touch with Dr. Pauli Kallio from the University of Turku. He was very excited about our project and eager to help. We were able to get Kallio’s groups plasmid maps from him and decided to use these as our starting material. As they had already tested this pathway, we could be sure that their genes were functional in E. coli.</p> | + | <p>Our propane pathway is based on the research done by <a href="http://www.nature.com/ncomms/2014/140902/ncomms5731/full/ncomms5731.html" target="_blank">Dr. Pauli Kallio <i>et al.</i></a> and <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61" target="_blank">Menon <i>et al.</i></a>. Right after we decided our topic we got in touch with Dr. Pauli Kallio from the University of Turku. He was very excited about our project and eager to help. We were able to get Kallio’s groups plasmid maps from him and decided to use these as our starting material. As they had already tested this pathway, we could be sure that their genes were functional in <i>E. coli</i>.</p> |
− | <p>Our chassis organism, <i>Escherichia coli</i> BL21 (DE3), was chosen because it’s a strain that produces the T7 promoter when induced with IPTG. This is the strain that was used in Kallio’s research and was available at our lab. In addition to the regular BL21 (DE3), Dr. Kallio was kind to send us a BL21(DE3) with YjgB and YqhD knocked out when we realized that producing some knock outs ourselves would be too expensive and time consuming. YjgB and YghD are E. | + | <p>Our chassis organism, <i>Escherichia coli</i> BL21 (DE3), was chosen because it’s a strain that produces the T7 promoter when induced with IPTG. This is the strain that was used in Kallio’s research and was available at our lab. In addition to the regular BL21 (DE3), Dr. Kallio was kind to send us a BL21(DE3) with YjgB and YqhD knocked out when we realized that producing some knock outs ourselves would be too expensive and time consuming. YjgB and YghD are <i>E. coli</i>’s endogenous genes which produce butyraldehyde consuming enzymes. This means that they compete with our pathway’s final enzyme, ADO, which uses butyraldehyde as its substrate.</p> |
<figure style="float:left;margin-right:20px;"> | <figure style="float:left;margin-right:20px;"> | ||
Line 121: | Line 121: | ||
We then moved forward to design our complete constructs. We used Kallio’s group’s constructs as a basis, and arranged the genes similarly. The arrangement of our first plasmid is the same as the original one. It starts with a T7 promoter and an additional lac operator. The T7 polymerase is IPTG inducible in our BL21 (DE3) strain, but we wanted to make sure there were no leaks in our system before the actual induction. That’s why the promoters adjacent to the genes are induced by IPTG. Following the promoter, we have the YciA, Sfp and CAR each with their own RBS and a T7 terminator. We chose to use the same RBS's and terminator sequences as Kallio's group, as they had already tested the system. Our construct's terminator sequence is used in common cloning vectors (such as pET and pDF). Our second plasmid includes the same promoter, RBS’s, and terminator, but the genes AtoB, Hbd, Crt and Ter, which is a similar construct as the original one made by Kallio's group. We decided to add another promoter to the plasmid to create two operon systems. We did this to ensure that all the genes would be transcribed by the polymerase. The last genes in our second construct, ADO, PetF and Fpr function under the same promoter and induction systems as all the other genes.</p> | We then moved forward to design our complete constructs. We used Kallio’s group’s constructs as a basis, and arranged the genes similarly. The arrangement of our first plasmid is the same as the original one. It starts with a T7 promoter and an additional lac operator. The T7 polymerase is IPTG inducible in our BL21 (DE3) strain, but we wanted to make sure there were no leaks in our system before the actual induction. That’s why the promoters adjacent to the genes are induced by IPTG. Following the promoter, we have the YciA, Sfp and CAR each with their own RBS and a T7 terminator. We chose to use the same RBS's and terminator sequences as Kallio's group, as they had already tested the system. Our construct's terminator sequence is used in common cloning vectors (such as pET and pDF). Our second plasmid includes the same promoter, RBS’s, and terminator, but the genes AtoB, Hbd, Crt and Ter, which is a similar construct as the original one made by Kallio's group. We decided to add another promoter to the plasmid to create two operon systems. We did this to ensure that all the genes would be transcribed by the polymerase. The last genes in our second construct, ADO, PetF and Fpr function under the same promoter and induction systems as all the other genes.</p> | ||
− | <p>Instead of Hbd, our Propane Plasmid 2 originally had FadB2 as the second gene. Hbd was originally used in Kallio's constructs, but FadB2 was used in an earlier experiment. Due to an error, FadB2 was built into our original construct instead of Hbd. According to literature, FadB2 had been shown to function in <i> E. coli </i>, but after the <a href="https://2015.igem.org/Team:Aalto-Helsinki/Kinetics">pathway's bottlenecks</a> were modeled, it became clear that the enzyme's kinetics properties were a serious problem. We then changed this gene to Hbd in our constructs, which functioned much better according to our model.</p> | + | <p>Instead of Hbd, our Propane Plasmid 2 originally had FadB2 as the second gene. Hbd was originally used in Kallio's constructs, but FadB2 was used in an earlier experiment. Due to an error, FadB2 was built into our original construct instead of Hbd. According to literature, FadB2 had been shown to function in <i>E. coli</i>, but after the <a href="https://2015.igem.org/Team:Aalto-Helsinki/Kinetics">pathway's bottlenecks</a> were modeled, it became clear that the enzyme's kinetics properties were a serious problem. We then changed this gene to Hbd in our constructs, which functioned much better according to our model.</p> |
<figure style="float:right;margin-left:20px;"> | <figure style="float:right;margin-left:20px;"> | ||
Line 130: | Line 130: | ||
<p>We first planned on creating a BioBrick from each of our genes and assembling the plasmids with the three antibiotics assembly method, but soon realized it would take too much time. We were then introduced to the Gibson Assembly system, and decided to give it a try. This is also when we decided to provide whole plasmids that would allow easy production of propane, rather than creating a separate brick of each of our genes.</p> | <p>We first planned on creating a BioBrick from each of our genes and assembling the plasmids with the three antibiotics assembly method, but soon realized it would take too much time. We were then introduced to the Gibson Assembly system, and decided to give it a try. This is also when we decided to provide whole plasmids that would allow easy production of propane, rather than creating a separate brick of each of our genes.</p> | ||
− | <p>So, we divided our whole plasmids into pieces of about 2000 bp each and included 30 bp overlaps into our gBlocks-to-be. This is when we also realized that synthesis isn’t as simple as it sounds: we had to optimize more than half of our sequences to be fit for gBlock synthesis. We will then proceed with <a href="https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix" target="_blank">NEBuilder kit</a>, <a href="http://nar.oxfordjournals.org/content/32/2/e19.full" target="_blank">overlapping PCR</a> (OE-PCR) and/or <a href="http://link.springer.com/article/10.1007%2Fs12033-014-9817-2#page-1" target="_blank">Exonuclease and Ligation-Independent Cloning</a> (ELIC) to combine our constructs. We used multiple methods because of the time constraint and the fact that we didn't know which one would work the best. Due to a construct design error our assembly pieces contained the BioBrick prefix in the very first part, but the last part did not include the suffix. The suffix was added by PCR after synthesis and the prefix & suffix areas functioned as the homologous overlap areas between our brick and the backbone.</p> | + | <p>So, we divided our whole plasmids into pieces of about 2000 bp each and included 30 bp overlaps into our gBlocks-to-be. This is when we also realized that synthesis isn’t as simple as it sounds: we had to optimize more than half of our sequences to be fit for gBlock synthesis. We will then proceed with <a href="https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix" target="_blank" style="padding:0">NEBuilder kit</a>, <a href="http://nar.oxfordjournals.org/content/32/2/e19.full" target="_blank">overlapping PCR</a> (OE-PCR) and/or <a href="http://link.springer.com/article/10.1007%2Fs12033-014-9817-2#page-1" target="_blank">Exonuclease and Ligation-Independent Cloning</a> (ELIC) to combine our constructs. We used multiple methods because of the time constraint and the fact that we didn't know which one would work the best. Due to a construct design error our assembly pieces contained the BioBrick prefix in the very first part, but the last part did not include the suffix. The suffix was added by PCR after synthesis and the prefix & suffix areas functioned as the homologous overlap areas between our brick and the backbone.</p> |
<figure style="float:left;margin-right:20px;"> | <figure style="float:left;margin-right:20px;"> | ||
Line 137: | Line 137: | ||
</figure> | </figure> | ||
− | <p>In the assembly phase, we also had to take into account the plasmids we were to use. As our whole system would require three plasmids altogether if we were to add the cellulose hydrolysing enzymes to the same bacteria, we had to be careful about the plasmids’ compatibility groups. Our plasmids needed different antibiotic resistances and intercompatible origins of replication. After our constructs were successfully assembled they were sent for sequencing to check that everything worked as expected. We then transformed them into competent E. coli BL21(DE3) ΔyjgB ΔyqhD strain with chemical transformation and screened the transformants with double-antibiotic plates. These cells should be able to produce propane. All we would need to do is induce the production with IPTG and identify the propane by gas chromatography.</p> | + | <p>In the assembly phase, we also had to take into account the plasmids we were to use. As our whole system would require three plasmids altogether if we were to add the cellulose hydrolysing enzymes to the same bacteria, we had to be careful about the plasmids’ compatibility groups. Our plasmids needed different antibiotic resistances and intercompatible origins of replication. After our constructs were successfully assembled they were sent for sequencing to check that everything worked as expected. We then transformed them into competent <i>E. coli</i> BL21(DE3) ΔyjgB ΔyqhD strain with chemical transformation and screened the transformants with double-antibiotic plates. These cells should be able to produce propane. All we would need to do is induce the production with IPTG and identify the propane by gas chromatography.</p> |
</section> | </section> | ||
Line 162: | Line 162: | ||
<figcaption><center><b>Figure 5.</b> Cellulose Plasmid</center></figcaption> | <figcaption><center><b>Figure 5.</b> Cellulose Plasmid</center></figcaption> | ||
</figure> | </figure> | ||
− | <p><i>E.coli</i> strains do not naturally contain any secretion systems for endoglucanases and exoglucanases so if the bacteria has previously been researched for cellulose degradation, usually the enzymes just overflow from cytoplasm to environment when enough proteins are produced. However, BL21 strain which we were using naturally contained β-glucosidase gene in the genome but because the expression levels and enzyme activities were poorly documented in databases, we decided to use the same gene <a href="https://2010.igem.org/Team:Osaka" target="_blank">Osaka 2010 team</a> was using from Distribution Kit. For cenA | + | <p><i>E.coli</i> strains do not naturally contain any secretion systems for endoglucanases and exoglucanases so if the bacteria has previously been researched for cellulose degradation, usually the enzymes just overflow from cytoplasm to environment when enough proteins are produced. However, BL21 strain which we were using naturally contained β-glucosidase gene in the genome but because the expression levels and enzyme activities were poorly documented in databases, we decided to use the same gene <a href="https://2010.igem.org/Team:Osaka" target="_blank">Osaka 2010 team</a> was using from Distribution Kit. For cenA, cex, and β-glucosidase secretion, pelB-secretion tag sequence <a href="http://parts.igem.org/Part:BBa_J32015">BBa_J32015</a> made by 2010 Duke team was synthesized prior to the coding sequences of the cellulases which would then move into cell’s periplasmic space.</p> |
− | <p>Cellulase producing capacity would | + | <p>Cellulase producing capacity would have been first investigated with carboxymethyl cellulose (CMC) plates which are labeled with Congo Red assay. Congo red dye is the sodium salt of 3,3'-([1,1'-biphenyl]-4,4'-diyl)bis(4-aminonaphthalene-1-sulfonic acid) which has a strong affinity to cellulose fibers. If the cellulose polymers are digested by enzymes on the plate, the spot will be changed into colorless and a halo will appear around the bacterial colony. However, the method will not tell whether glucose is produced or not.</p> |
− | <p>For glucose analysis, 3,5-dinitrosalicylic acid would | + | <p>For glucose analysis, 3,5-dinitrosalicylic acid would have been utilized. It is a compound which reacts with reductive sugars like glucose forming 3-amino-5-nitrosalicylic acid. The product absorbs light with the wavelength of 540 nm. Three different controls are needed when analyzing the produced glucose concentration because the cultivation liquid with CMC already contains sugar: one without any strain, one with <i>E.coli</i> without cellulase producing capacity and one with cellulose hydrolyzing. Furthermore, liquid chromatography could also be utilized if we find proper equipment.</p> |
Line 178: | Line 178: | ||
<h2>Micelle Fusions Enhancing the Production</h2> | <h2>Micelle Fusions Enhancing the Production</h2> | ||
− | <p>Based on the previous <a href="http://www.nature.com/ncomms/2014/140902/ncomms5731/full/ncomms5731.html" target="_blank">studies</a> about this <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61">pathway </a>, we knew the propane yields weren’t very high. We thought about trying to enhance the system by searching for homologs for the enzymes, but thought this would be too time-consuming and also not very innovative. We then ran into a research article by <a href="http://www.nature.com/nmat/journal/v14/n1/full/nmat4118.html" target="_blank">Huber <i>et al.</i></a>. The group had designed a synthetic amphiphilic protein that spontaneously formed membrane-like structures inside the cell. These proteins were designed quite like membrane lipids: there is a hydrophilic and a hydrophobic end. According to the energy minimum principle, the proteins’ hydrophilic ends will face the liquid phase of the cell and the hydrophobic ends will pack together. This way the proteins will be able to form either a double layered vesicle (similar to the double lipid layer) or a micelle.</p> | + | <p>Based on the previous <a href="http://www.nature.com/ncomms/2014/140902/ncomms5731/full/ncomms5731.html" target="_blank">studies</a> about this <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61">pathway</a>, we knew the propane yields weren’t very high. We thought about trying to enhance the system by searching for homologs for the enzymes, but thought this would be too time-consuming and also not very innovative. We then ran into a research article by <a href="http://www.nature.com/nmat/journal/v14/n1/full/nmat4118.html" target="_blank">Huber <i>et al.</i></a>. The group had designed a synthetic amphiphilic protein that spontaneously formed membrane-like structures inside the cell. These proteins were designed quite like membrane lipids: there is a hydrophilic and a hydrophobic end. According to the energy minimum principle, the proteins’ hydrophilic ends will face the liquid phase of the cell and the hydrophobic ends will pack together. This way the proteins will be able to form either a double layered vesicle (similar to the double lipid layer) or a micelle.</p> |
<figure style="float:left;margin-right:20px;"> | <figure style="float:left;margin-right:20px;"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/e/e4/Aalto-Helsinki_micelle_circle_approach_2.png" style="width:200px;"/> |
<figcaption></figcaption> | <figcaption></figcaption> | ||
</figure> | </figure> | ||
Line 256: | Line 256: | ||
<figure style="float:right"> | <figure style="float:right"> | ||
<img src="https://static.igem.org/mediawiki/2015/f/f5/Aalto-Helsinki_continuous_reactor.png" style="width:160px;"/> | <img src="https://static.igem.org/mediawiki/2015/f/f5/Aalto-Helsinki_continuous_reactor.png" style="width:160px;"/> | ||
− | <figcaption></figcaption> | + | <figcaption><center><b>Figure 10.</b>Chemostat reactor </br> with feed- and outline</center></figcaption> |
</figure> | </figure> | ||
− | <p>Because the strain BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) produces propane only by inducing with IPTG instead of constitutive production, certain limitations are met when designing the chemostat conditions. Cultivating microbes with a maximum capacity would consume too much IPTG which is quite a valuable reagent, as a diluting rate needs to be high. Therefore, instead of exploring the | + | <p>Because the strain BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) produces propane only by inducing with IPTG instead of constitutive production, certain limitations are met when designing the chemostat conditions. Cultivating microbes with a maximum capacity would consume too much IPTG which is quite a valuable reagent, as a diluting rate needs to be high. Therefore, instead of exploring the maximum yield by adding abundantly IPTG, the chemostat will be used for investigating how the environmental conditions affect the product formation. The most simple way to do this is to wait until the steady state is achieved without any induction and then add IPTG. To see how change of process conditions affects the cell density, OD600 is measured all the time.</p> |
− | <p style="margin-bottom:0;padding-bottom:10%";>The overall media volume of chemostat will be 500 ml due to the limit of the IPTG amount. The steady state is approximated to take 10 hours but it may vary radically. Diluting rate is kept to 0,072 1/h and glucose feed 20 g/L. When OD600 reaches its balance, | + | <p style="margin-bottom:0;padding-bottom:10%";>The overall media volume of chemostat will be 500 ml due to the limit of the IPTG amount. The steady state is approximated to take 10 hours but it may vary radically. Diluting rate is kept to 0,072 1/h and glucose feed 20 g/L. When OD600 reaches its balance, IPTG is added to the media to a concentration of 2,0 mM. Induction will take another 10 hours so the overall process time will be probably few days. Gas products will be measured with GC-MS analysator which will be connected with the outlet stream of the chemostat. Because the reactor size is limited (2 L), only few sensors can be attached to the same unit. We will measure the cell density, pH, dissolved oxygen and composition of outstream gas.</p> |
Latest revision as of 04:23, 29 October 2015