Difference between revisions of "Team:Stanford-Brown/SB16 BioMembrane Nylon"
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| + | <div class="col-sm-12 pagetext-L"><div class="text">Nylon is a designation for a family of semi-aromatic polyamids that can be worked into various useful forms. Thin films of nylon have similar properties to polyvinyl chloride plastic wrap, capable of stretching under stress before tearing. Nylon fibers are lightweight and have high tensile strengths, making them very useful when woven into fabrics. Besides its applications in clothing, woven nylon is most commonly used in hot air balloon membranes and sailboat sails due to its durability. Properties of nylon materials such as tensile strength, heat resistance, and abrasion resistance can be increased by crosslinking the polymers using various methods. [1] The most common forms of nylon in the textile and plastic industries are nylon-6 and nylon-6,6, both of which have similar physical characteristics. While nylon-6,6 is a copolymer, nylon-6 is a homopolymer, making it a more appropriate candidate for biosynthesis. For these reasons, our team identified nylon-6 as one of our candidate biomembranes. | ||
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| − | <div class="col-sm-12 pagetext-L"><div class="text">Nylon-6 is industrially produced through the chain-growth polymerization of caprolactam, the cyclical form of 6-aminocaproic acid (6-ACA). Our team investigated potential biosynthesis routes for this monomer, looking for pathways with common starting substrates to design a production system for 6-ACA that could be implemented on an extraterrestrial body. Past retrosynthetic analyses have uncovered two fermentative pathways for the production of 6-ACA, which both lacked required biocatalytic steps until 2015. [ | + | <div class="col-sm-12 pagetext-L"><div class="text">Nylon-6 is industrially produced through the chain-growth polymerization of caprolactam, the cyclical form of 6-aminocaproic acid (6-ACA). Our team investigated potential biosynthesis routes for this monomer, looking for pathways with common starting substrates to design a production system for 6-ACA that could be implemented on an extraterrestrial body. Past retrosynthetic analyses have uncovered two fermentative pathways for the production of 6-ACA, which both lacked required biocatalytic steps until 2015. [2][3] Last October, researcher Stefan Turk published a study detailing and potential candidates enzymes for these steps and characterizing their activity in vivo in <i>E. coli.</i> [4] With the novel nature of his work in mind, our team contacted Turk in request of a sample of his engineered strain eAKP-672 with the highest 6-ACA yield when grown in glucose containing medium. We hoped to adjust Turk's synthetic plasmid designs and/or make our own genomic edits to optimize the metabolic flux of his biosynthetic pathways. Turk replied in the affirmative, shipping us two separate cell samples containing plasmids pAKP-96 and pAKP-444, which held the six enzymes necessary to induce one of the two above mentioned pathways in <i>Escherichia coli.</i> Unfortunately, issues with a large number of illegal restriction sites in the relevant genes made biobricking intractable, and we had to abandon this part of the project. |
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| + | <h1 class="sectionTitle-L firstTitle">Starting from Lysine</h1> | ||
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| + | <div class="col-sm-12 pagetext-L"><div class="text">Fortunately, 6-aminocaproic acid is only a single functional group away from the amino acid L-lysine. A great deal more detail can be found on the characterization page for the relevant biobrick, <a href="http://parts.igem.org/Part:BBa_K2027000">BBa_K2027000</a>. In short, we relied on the work of Tani et al. who discovered that an apoptosis-inducing protein with L-lysine-α-oxidase activity from <em>Scomber japonicus</em> retained that activity in a recombinant variant produced in bacteria.<sup>5</sup> We verified production, purification, and functionality of this enzyme, marking the first step in a potential semi-synthetic route to 6-aminocaproic acid, caprolactam, and nylon. One of the primary advantages of enzyme use is specific targeting, and this product successfully targets and changes the α-amino group of L-lysine, opening the door to less specific chemical methods of reduction that have been developed for decades. Of course, the possibility of utilizing additional enzymes in a search for yet another completely biological route is also open. We hope that by providing this functional enzyme to the registry in a form that is easily produced in high functional yield and purified, the next steps in this journey will become that much easier. | ||
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<div class="col-sm-12 pagetext-L"><div class="text"><i>References</i><br> | <div class="col-sm-12 pagetext-L"><div class="text"><i>References</i><br> | ||
| − | 1. https://www.google.com/patents/US20140134681< | + | 1. http://www.ptonline.com/articles/radiation-crosslinking-boosts-nylon-properties<br> |
| − | + | 2. https://www.google.com/patents/US20140134681<br> | |
| − | + | 3. https://www.google.com/patents/US20110091944<br> | |
| + | 4. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00129?journalCode<br> | ||
| + | 5. Tani, Y., Miyake, R., Yukami, R. et al. Appl Microbiol Biotechnol (2015) 99: 5045. doi:10.1007/s00253-014-6308-0<br> | ||
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Latest revision as of 03:58, 20 October 2016
Nylon-6 Biosynthesis
Nylon is a designation for a family of semi-aromatic polyamids that can be worked into various useful forms. Thin films of nylon have similar properties to polyvinyl chloride plastic wrap, capable of stretching under stress before tearing. Nylon fibers are lightweight and have high tensile strengths, making them very useful when woven into fabrics. Besides its applications in clothing, woven nylon is most commonly used in hot air balloon membranes and sailboat sails due to its durability. Properties of nylon materials such as tensile strength, heat resistance, and abrasion resistance can be increased by crosslinking the polymers using various methods. [1] The most common forms of nylon in the textile and plastic industries are nylon-6 and nylon-6,6, both of which have similar physical characteristics. While nylon-6,6 is a copolymer, nylon-6 is a homopolymer, making it a more appropriate candidate for biosynthesis. For these reasons, our team identified nylon-6 as one of our candidate biomembranes.
Nylon-6 is industrially produced through the chain-growth polymerization of caprolactam, the cyclical form of 6-aminocaproic acid (6-ACA). Our team investigated potential biosynthesis routes for this monomer, looking for pathways with common starting substrates to design a production system for 6-ACA that could be implemented on an extraterrestrial body. Past retrosynthetic analyses have uncovered two fermentative pathways for the production of 6-ACA, which both lacked required biocatalytic steps until 2015. [2][3] Last October, researcher Stefan Turk published a study detailing and potential candidates enzymes for these steps and characterizing their activity in vivo in E. coli. [4] With the novel nature of his work in mind, our team contacted Turk in request of a sample of his engineered strain eAKP-672 with the highest 6-ACA yield when grown in glucose containing medium. We hoped to adjust Turk's synthetic plasmid designs and/or make our own genomic edits to optimize the metabolic flux of his biosynthetic pathways. Turk replied in the affirmative, shipping us two separate cell samples containing plasmids pAKP-96 and pAKP-444, which held the six enzymes necessary to induce one of the two above mentioned pathways in Escherichia coli. Unfortunately, issues with a large number of illegal restriction sites in the relevant genes made biobricking intractable, and we had to abandon this part of the project.
Starting from Lysine
Fortunately, 6-aminocaproic acid is only a single functional group away from the amino acid L-lysine. A great deal more detail can be found on the characterization page for the relevant biobrick, BBa_K2027000. In short, we relied on the work of Tani et al. who discovered that an apoptosis-inducing protein with L-lysine-α-oxidase activity from Scomber japonicus retained that activity in a recombinant variant produced in bacteria.5 We verified production, purification, and functionality of this enzyme, marking the first step in a potential semi-synthetic route to 6-aminocaproic acid, caprolactam, and nylon. One of the primary advantages of enzyme use is specific targeting, and this product successfully targets and changes the α-amino group of L-lysine, opening the door to less specific chemical methods of reduction that have been developed for decades. Of course, the possibility of utilizing additional enzymes in a search for yet another completely biological route is also open. We hope that by providing this functional enzyme to the registry in a form that is easily produced in high functional yield and purified, the next steps in this journey will become that much easier.
References
1. http://www.ptonline.com/articles/radiation-crosslinking-boosts-nylon-properties
2. https://www.google.com/patents/US20140134681
3. https://www.google.com/patents/US20110091944
4. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00129?journalCode
5. Tani, Y., Miyake, R., Yukami, R. et al. Appl Microbiol Biotechnol (2015) 99: 5045. doi:10.1007/s00253-014-6308-0
1. http://www.ptonline.com/articles/radiation-crosslinking-boosts-nylon-properties
2. https://www.google.com/patents/US20140134681
3. https://www.google.com/patents/US20110091944
4. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00129?journalCode
5. Tani, Y., Miyake, R., Yukami, R. et al. Appl Microbiol Biotechnol (2015) 99: 5045. doi:10.1007/s00253-014-6308-0
