Difference between revisions of "Team:Aalto-Helsinki/Project"
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<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> |
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<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> <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). | + | <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> |
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− | <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;"> | ||
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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> |
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− | <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> | ||
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<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>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 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 E.coli without cellulase producing capacity and one with cellulose hydrolyzing. Furthermore, liquid chromatography could also be utilized if we find proper equipment.</p> | + | <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> |
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− | <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> | ||
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Latest revision as of 04:23, 29 October 2015