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| − | <h1>E. coli Producing Propane</h1>
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| − | <p>Our propane pathway is based on the research done by Pauli Kallio et al [<a href="http://www.nature.com/ncomms/2014/140902/ncomms5731/full/ncomms5731.html">1</a>] and Menon et al [<a href="http://www.biotechnologyforbiofuels.com/content/8/1/61">2</a>]. Right in the beginning we got in touch with Pauli Kallio from the University of Turku. He was very excited about our project and eager to help. We were able to get Pauli’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>
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| − | <p>Our chassis organism, E. coli BL21 (DE3) was chosen because it’s a strain that produces 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), Pauli 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. coli’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>
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| − | <p>We had been warned about the pathway’s vulnerabilities: it consisted of 10 different enzymes and in earlier research, it had been built with a minimum of three different plasmids. The stress for the bacteria was high. We thought that one way of reducing this stress was to use just two plasmids for the propane pathway. We then moved forward to design our complete constructs. We used Pauli’s group’s construct 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 a separate rbs (Lähde???) and a T7 terminator. We chose to use the same RBS's and terminator sequences as Pauli Kallio's group, as they had already tested the system. Our constructs 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 an 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 were arranged quite randomly and function under the same promoter and induction systems as all the other genes.</p>
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| − | <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 gave up on the idea of creating a separate brick of each of our genes, but rather wanted to provide whole plasmids that would allow easy production of propane.</p>
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| − | <p>So, we divided our whole plasmids into pieces of about 2000bp each and included 30bp 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">NEBuilder kit</a>, <a href="http://nar.oxfordjournals.org/content/32/2/e19.full">overlapping PCR</a> (OE-PCR) and/or <a href="http://link.springer.com/article/10.1007%2Fs12033-014-9817-2#page-1">ELIC</a> to combine our constructs. We used multiple methods because of the time-constrait 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>
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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. We ended up using one <a href="http://parts.igem.org/Part:pSB6A1">plasmid backbone</a> from the iGEM distribution kit. Our second and third plasmids are commercial Novagen plasmids pACYCDuet-1 and pCDFDuet-1. <span style="color:red">Needs explanation of how we got rid of the extra T7 promoters!!</span> Based on our pathway kinetics model, we assmbled our constructs so that the most rate-limiting enzyme, ADO was in the highest copy-number plasmid and second bottleneck CAR in the backbone with the next highest copy-number. Unfortunately the construct design process was too far at this point for us to assemble both CAR and ADO into the high copy-number plasmid. 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>
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