Difference between revisions of "Team:Stanford-Brown/PHA"
| Line 119: | Line 119: | ||
<p>P(3HB) is the most common polyhydroxyalkanoate, which is a group of fully biodegradable polymers with properties similar to those of petroleum-based plastics. It can be produced in a variety of microorganisms, and its synthesis as a compound for energy storage in vivo has been extensively studied [1]. P(3HB) accumulates inside cells, and has been reported to occupy up to 90% (w/w) of the dry cell mass in certain species [2]. However, P(3HB) has yet to become integrated into the plastics market on Earth due to high costs of both fermentation and purification [3]. This summer, we focused on making the production, extraction, and use of P(3HB) more feasible on a lunar or Martian base.</p> | <p>P(3HB) is the most common polyhydroxyalkanoate, which is a group of fully biodegradable polymers with properties similar to those of petroleum-based plastics. It can be produced in a variety of microorganisms, and its synthesis as a compound for energy storage in vivo has been extensively studied [1]. P(3HB) accumulates inside cells, and has been reported to occupy up to 90% (w/w) of the dry cell mass in certain species [2]. However, P(3HB) has yet to become integrated into the plastics market on Earth due to high costs of both fermentation and purification [3]. This summer, we focused on making the production, extraction, and use of P(3HB) more feasible on a lunar or Martian base.</p> | ||
| − | <p>(1) Increasing P(3HB) Yield</p> | + | <p><b>(1) Increasing P(3HB) Yield</b></p> |
<p>Increasing the yield of P(3HB) from a cell culture would decrease the time needed to complete the plastic production and extraction process by making smaller cultures more efficient. We have used the phaCAB operon from Ralstonia eutropha H16 that encodes the three genes required for P(3HB) production: PhaA, PhaB, and PhaC. Previous iGEM teams (Tokyo Tech 2012 and Imperial 2013) were able to successfully produce P(3HB) in vivo using BioBricks that encode these three genes. The pathway for P(3HB) production is as follows. First, 3-ketothiolase, encoded by PhaA, combines two molecules of acetyl-CoA to form acetoacetyl-CoA. This is then reduced by acetoacetyl-CoA reductase, encoded by PhaB, to form (R)-3-hydroxybutyl-CoA. This is then polymerized by PHA synthase, encoded by PhaC, forming poly-3-hydroxybuterate.</p> | <p>Increasing the yield of P(3HB) from a cell culture would decrease the time needed to complete the plastic production and extraction process by making smaller cultures more efficient. We have used the phaCAB operon from Ralstonia eutropha H16 that encodes the three genes required for P(3HB) production: PhaA, PhaB, and PhaC. Previous iGEM teams (Tokyo Tech 2012 and Imperial 2013) were able to successfully produce P(3HB) in vivo using BioBricks that encode these three genes. The pathway for P(3HB) production is as follows. First, 3-ketothiolase, encoded by PhaA, combines two molecules of acetyl-CoA to form acetoacetyl-CoA. This is then reduced by acetoacetyl-CoA reductase, encoded by PhaB, to form (R)-3-hydroxybutyl-CoA. This is then polymerized by PHA synthase, encoded by PhaC, forming poly-3-hydroxybuterate.</p> | ||
| Line 126: | Line 126: | ||
<p>Coenzyme A biosynthesis is a five-step process that is primarily regulated by the first enzyme in the pathway, pantothenate kinase (encoded by the gene PanK, which is also called CoaA) [4]. Pantothenate kinase stringently controls amount of coenzyme A produced in E. coli because the pantothenate kinase produced by E. coli is significantly inhibited by coenzyme A and its thioesters (such as acetyl-coA). Therefore, since we wanted to increase coenzyme A synthesis, we had to find a way to get around this negative feedback inhibition. Fortunately, the pantothenate kinase made in Staphylococcus aureus does not experience feedback inhibition from coenzyme A or its thioesters [5]. Indeed, this allows S. aureus to accumulate high levels of coenzyme A. We ordered the S. aureus pantothenate kinase sequence from IDT and inserted into the backbone PSB1C3 in front of the phaCAB operon designed by the Tokyo Tech iGEM team in 2012 with the hybrid promoter designed by the Imperial College iGEM team in 2013. The results section below shows that inserting the gene for pantothenate kinase allows for higher levels of P(3HB) production in E. coli.</p> | <p>Coenzyme A biosynthesis is a five-step process that is primarily regulated by the first enzyme in the pathway, pantothenate kinase (encoded by the gene PanK, which is also called CoaA) [4]. Pantothenate kinase stringently controls amount of coenzyme A produced in E. coli because the pantothenate kinase produced by E. coli is significantly inhibited by coenzyme A and its thioesters (such as acetyl-coA). Therefore, since we wanted to increase coenzyme A synthesis, we had to find a way to get around this negative feedback inhibition. Fortunately, the pantothenate kinase made in Staphylococcus aureus does not experience feedback inhibition from coenzyme A or its thioesters [5]. Indeed, this allows S. aureus to accumulate high levels of coenzyme A. We ordered the S. aureus pantothenate kinase sequence from IDT and inserted into the backbone PSB1C3 in front of the phaCAB operon designed by the Tokyo Tech iGEM team in 2012 with the hybrid promoter designed by the Imperial College iGEM team in 2013. The results section below shows that inserting the gene for pantothenate kinase allows for higher levels of P(3HB) production in E. coli.</p> | ||
| + | |||
| + | <p><b>(2) Facilitating P(3HB) Extraction</b></p> | ||
| + | |||
| + | <p>Since bacteria that naturally synthesize P(3HB) use the plastic as an energy storage mechanism, the plastic is produced inside the cell and stays there. Currently, the isolation of P(3HB) from the cells and culture media is one of the main bottlenecks in the P(3HB) production process – and the main source of its high cost of approximately $4-6 USD [6]. Traditional isolation methods include the use of large amounts of chemicals that can be hazardous to human health and the environment, such as chloroform and sodium hypochlorite, or other problems such as large volumes of waste water produced or impurities in the polymers [6]. Since one of our team’s goals has been to reduce up-mass of materials on space flights, we wanted to minimize the amounts of harmful (and heavy) chemicals that would need to be brought up as payload to extract the P(3HB).</p> | ||
| + | |||
| + | <p>We accomplished this by introducing a lysis device into the PSB1C3 plasmid that contained the phaCAB operon and the gene for pantothenate kinase. The lysis device was developed by the 2008 Berkeley iGEM team. The device contains T7 phage antiholin under a weak promoter, and T7 phage holin and endolysin inducible under (L)-arabinose. This lysis device was not reported to kill entire populations of E. coli, and we saw this as an advantage for a continuous plastic-production system. We would be able to cause part of the population to lyse and release its P(3HB) into the media. The rest of the population would continue growing and producing plastic. The plastic can be recovered from the media by causing the granules to aggregate, and then requiring lower amounts of solvents and less time to complete the purification process [7]. The results section below demonstrates the success of the lysis system and compares the efficiency of the recovery methods with and without the release of the P(3HB) into the media.</p> | ||
| Line 208: | Line 214: | ||
<p>[4] Leonardi, R. et al. Coenzyme A: Back in action. Progress in Lipid Research 44, 2005. 125 – 153.</p> | <p>[4] Leonardi, R. et al. Coenzyme A: Back in action. Progress in Lipid Research 44, 2005. 125 – 153.</p> | ||
| − | <p>[5] Leonardi, R. et al. A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A. The Journal of Biological Chemistry 280, 2005. 3312 – 3322. | + | <p>[5] Leonardi, R. et al. A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A. The Journal of Biological Chemistry 280, 2005. 3312 – 3322.</p> |
| + | |||
| + | <p>[6] Jacquel, N. et al. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering Journal 39 (1), 2008. 15 – 27.</p> | ||
| + | |||
| + | <p>[7] Rahman, A. et al. Secretion of polyhydroxybuterate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering 7 (24), 2013.</p> | ||
| + | |||
<p> | <p> | ||
Revision as of 06:21, 9 September 2015
P(3HB) In vivo synthesis of biodegradable polymers
Abstract
We produced poly-(3-hydroxybutyrate) [P(3HB)] as a substrate to fold biOrigami. P(3HB) is a biopolymer with thermoplastic properties, meaning that it contracts in heat. Unlike polystyrene, P(3HB) is biodegradable. Building on previous iGEM teams’ work on P(3HB), we have contributed BioBricks that increase P(3HB) production and facilitate its extraction from Escherichia coli, and have processed the polymer into useful sheets. See our biobricks here:
See our BioBricksIntroduction and Background
We are using recombinant E. coli to produce P(3HB) for folding biOrigami. Since plastics on earth have deleterious environmental impacts, we wanted to focus on producing one biodegradable plastic that could be synthesized for use on the moon or on Mars.
P(3HB) is the most common polyhydroxyalkanoate, which is a group of fully biodegradable polymers with properties similar to those of petroleum-based plastics. It can be produced in a variety of microorganisms, and its synthesis as a compound for energy storage in vivo has been extensively studied [1]. P(3HB) accumulates inside cells, and has been reported to occupy up to 90% (w/w) of the dry cell mass in certain species [2]. However, P(3HB) has yet to become integrated into the plastics market on Earth due to high costs of both fermentation and purification [3]. This summer, we focused on making the production, extraction, and use of P(3HB) more feasible on a lunar or Martian base.
(1) Increasing P(3HB) Yield
Increasing the yield of P(3HB) from a cell culture would decrease the time needed to complete the plastic production and extraction process by making smaller cultures more efficient. We have used the phaCAB operon from Ralstonia eutropha H16 that encodes the three genes required for P(3HB) production: PhaA, PhaB, and PhaC. Previous iGEM teams (Tokyo Tech 2012 and Imperial 2013) were able to successfully produce P(3HB) in vivo using BioBricks that encode these three genes. The pathway for P(3HB) production is as follows. First, 3-ketothiolase, encoded by PhaA, combines two molecules of acetyl-CoA to form acetoacetyl-CoA. This is then reduced by acetoacetyl-CoA reductase, encoded by PhaB, to form (R)-3-hydroxybutyl-CoA. This is then polymerized by PHA synthase, encoded by PhaC, forming poly-3-hydroxybuterate.
To increase the yield of P(3HB), we looked for a way to increase the amount of acetyl-coA, the precursor molecule to P(3HB) formation. Acetyl-CoA is composed of an acetyl group bound to coenzyme A. Coenzyme A consists of a beta-mercaptoethylamine group linked to pantothenic acid. On the Tokyo Tech 2012 iGEM team’s wiki, their results showed that adding pantothenic acid into their culture media led to increased yields of P(3HB). Therefore, we decided to increase the production of coenzyme A, which would then allow the cells to synthesize greater amounts of P(3HB).
Coenzyme A biosynthesis is a five-step process that is primarily regulated by the first enzyme in the pathway, pantothenate kinase (encoded by the gene PanK, which is also called CoaA) [4]. Pantothenate kinase stringently controls amount of coenzyme A produced in E. coli because the pantothenate kinase produced by E. coli is significantly inhibited by coenzyme A and its thioesters (such as acetyl-coA). Therefore, since we wanted to increase coenzyme A synthesis, we had to find a way to get around this negative feedback inhibition. Fortunately, the pantothenate kinase made in Staphylococcus aureus does not experience feedback inhibition from coenzyme A or its thioesters [5]. Indeed, this allows S. aureus to accumulate high levels of coenzyme A. We ordered the S. aureus pantothenate kinase sequence from IDT and inserted into the backbone PSB1C3 in front of the phaCAB operon designed by the Tokyo Tech iGEM team in 2012 with the hybrid promoter designed by the Imperial College iGEM team in 2013. The results section below shows that inserting the gene for pantothenate kinase allows for higher levels of P(3HB) production in E. coli.
(2) Facilitating P(3HB) Extraction
Since bacteria that naturally synthesize P(3HB) use the plastic as an energy storage mechanism, the plastic is produced inside the cell and stays there. Currently, the isolation of P(3HB) from the cells and culture media is one of the main bottlenecks in the P(3HB) production process – and the main source of its high cost of approximately $4-6 USD [6]. Traditional isolation methods include the use of large amounts of chemicals that can be hazardous to human health and the environment, such as chloroform and sodium hypochlorite, or other problems such as large volumes of waste water produced or impurities in the polymers [6]. Since one of our team’s goals has been to reduce up-mass of materials on space flights, we wanted to minimize the amounts of harmful (and heavy) chemicals that would need to be brought up as payload to extract the P(3HB).
We accomplished this by introducing a lysis device into the PSB1C3 plasmid that contained the phaCAB operon and the gene for pantothenate kinase. The lysis device was developed by the 2008 Berkeley iGEM team. The device contains T7 phage antiholin under a weak promoter, and T7 phage holin and endolysin inducible under (L)-arabinose. This lysis device was not reported to kill entire populations of E. coli, and we saw this as an advantage for a continuous plastic-production system. We would be able to cause part of the population to lyse and release its P(3HB) into the media. The rest of the population would continue growing and producing plastic. The plastic can be recovered from the media by causing the granules to aggregate, and then requiring lower amounts of solvents and less time to complete the purification process [7]. The results section below demonstrates the success of the lysis system and compares the efficiency of the recovery methods with and without the release of the P(3HB) into the media.
Experiment Engineering E. coli to produce polystyrene
Donec ullamcorper nulla non metus auctor fringilla. Vestibulum id ligula porta felis euismod semper. Praesent commodo cursus magna, vel scelerisque nisl consectetur. Fusce dapibus, tellus ac cursus commodo.
Donec tincidunt aliquet justo, sit amet mollis purus varius ac. Quisque ac sapien eu ante convallis cursus congue vel odio. Sed efficitur sapien ut eros sodales ornare. Vestibulum pellentesque lorem sed nulla interdum, non tincidunt velit sagittis. Vestibulum cursus, enim eu porta euismod, enim lectus facilisis diam, at sodales metus ligula sit amet eros. Sed ullamcorper, mauris nec mollis pretium, justo ligula dapibus nulla, non elementum nisl libero ut elit. Proin mi urna, finibus at scelerisque quis, porttitor at mauris. Nulla laoreet venenatis cursus. Vivamus et pellentesque quam, eget malesuada ex. Quisque eu massa ligula. Nam interdum dui sed laoreet efficitur. Aliquam sed vulputate orci. Pellentesque sed sollicitudin lectus. Vivamus nec tortor risus. Vestibulum malesuada feugiat lorem a dignissim. In diam mauris, venenatis at vulputate eget, venenatis sit amet metus. Suspendisse ut mi in ipsum sagittis malesuada at nec erat. Etiam volutpat risus quis nisi hendrerit porttitor vel eu tortor. Donec venenatis, risus sit amet ullamcorper scelerisque, tellus erat consequat nibh, vel dictum velit augue id leo. In eleifend tristique ipsum sed dignissim. Duis mattis, ipsum nec aliquet varius, turpis orci tempus nulla, in sodales libero massa at diam. Nulla maximus eros sed venenatis congue. Phasellus diam nunc, ullamcorper vitae tempor eget, sagittis eu odio. Praesent a mauris porttitor, mattis sem a, sodales massa. Proin et justo lectus. Proin varius magna ac leo ullamcorper accumsan. Proin id diam eget dolor vulputate mattis. Suspendisse pellentesque, nunc sit amet blandit feugiat, risus eros egestas massa, nec condimentum ante sapien ac velit. Vivamus efficitur justo dolor, at gravida lorem venenatis at. Aenean at ligula sapien. Mauris eget eleifend justo, eget faucibus ante. Ut mattis ante vitae dignissim maximus. Integer feugiat arcu purus, a viverra dui elementum vitae. Phasellus mattis porttitor iaculis. In eu nisi eu augue lacinia fringilla venenatis at nunc. Nam est erat, hendrerit ac dignissim sed, mollis eu eros. Morbi vel egestas dui, consectetur posuere nisi. Aliquam vitae tortor vulputate, fringilla est vel, faucibus diam. Suspendisse potenti. Donec sed commodo nulla. Duis feugiat, diam eu pulvinar rhoncus, arcu erat pretium orci, ut porta diam elit eu mi. Etiam eros massa, egestas eu mattis id, hendrerit at ligula. Duis placerat felis nec risus volutpat lobortis. Sed elementum, dolor non feugiat placerat, libero sapien pharetra diam, sed faucibus est ex tristique sem. Vivamus rutrum libero eget mollis sodales. Pellentesque vel scelerisque felis, a imperdiet erat. Fusce quis nisl magna. Sed non libero ultrices sapien hendrerit suscipit aliquet convallis leo. Quisque nec aliquam libero, in commodo ex. In eget nulla consequat, commodo quam id, hendrerit velit. Vestibulum non interdum enim. Ut elit justo, suscipit vel pretium vitae, rutrum sed dui. Donec vehicula sit amet ex ac finibus. Donec ultrices tellus et laoreet dictum.
Data and Results Optimizing the production of biological PHA
Donec ullamcorper nulla non metus auctor fringilla. Vestibulum id ligula porta felis euismod semper. Praesent commodo cursus magna, vel scelerisque nisl consectetur. Fusce dapibus, tellus ac cursus commodo.
Donec tincidunt aliquet justo, sit amet mollis purus varius ac. Quisque ac sapien eu ante convallis cursus congue vel odio. Sed efficitur sapien ut eros sodales ornare. Vestibulum pellentesque lorem sed nulla interdum, non tincidunt velit sagittis. Vestibulum cursus, enim eu porta euismod, enim lectus facilisis diam, at sodales metus ligula sit amet eros. Sed ullamcorper, mauris nec mollis pretium, justo ligula dapibus nulla, non elementum nisl libero ut elit. Proin mi urna, finibus at scelerisque quis, porttitor at mauris. Nulla laoreet venenatis cursus. Vivamus et pellentesque quam, eget malesuada ex. Quisque eu massa ligula. Nam interdum dui sed laoreet efficitur. Aliquam sed vulputate orci. Pellentesque sed sollicitudin lectus. Vivamus nec tortor risus. Vestibulum malesuada feugiat lorem a dignissim. In diam mauris, venenatis at vulputate eget, venenatis sit amet metus. Suspendisse ut mi in ipsum sagittis malesuada at nec erat. Etiam volutpat risus quis nisi hendrerit porttitor vel eu tortor. Donec venenatis, risus sit amet ullamcorper scelerisque, tellus erat consequat nibh, vel dictum velit augue id leo. In eleifend tristique ipsum sed dignissim. Duis mattis, ipsum nec aliquet varius, turpis orci tempus nulla, in sodales libero massa at diam. Nulla maximus eros sed venenatis congue. Phasellus diam nunc, ullamcorper vitae tempor eget, sagittis eu odio. Praesent a mauris porttitor, mattis sem a, sodales massa. Proin et justo lectus. Proin varius magna ac leo ullamcorper accumsan. Proin id diam eget dolor vulputate mattis. Suspendisse pellentesque, nunc sit amet blandit feugiat, risus eros egestas massa, nec condimentum ante sapien ac velit. Vivamus efficitur justo dolor, at gravida lorem venenatis at. Aenean at ligula sapien. Mauris eget eleifend justo, eget faucibus ante. Ut mattis ante vitae dignissim maximus. Integer feugiat arcu purus, a viverra dui elementum vitae. Phasellus mattis porttitor iaculis. In eu nisi eu augue lacinia fringilla venenatis at nunc. Nam est erat, hendrerit ac dignissim sed, mollis eu eros. Morbi vel egestas dui, consectetur posuere nisi. Aliquam vitae tortor vulputate, fringilla est vel, faucibus diam. Suspendisse potenti. Donec sed commodo nulla. Duis feugiat, diam eu pulvinar rhoncus, arcu erat pretium orci, ut porta diam elit eu mi. Etiam eros massa, egestas eu mattis id, hendrerit at ligula. Duis placerat felis nec risus volutpat lobortis. Sed elementum, dolor non feugiat placerat, libero sapien pharetra diam, sed faucibus est ex tristique sem. Vivamus rutrum libero eget mollis sodales. Pellentesque vel scelerisque felis, a imperdiet erat. Fusce quis nisl magna. Sed non libero ultrices sapien hendrerit suscipit aliquet convallis leo. Quisque nec aliquam libero, in commodo ex. In eget nulla consequat, commodo quam id, hendrerit velit. Vestibulum non interdum enim. Ut elit justo, suscipit vel pretium vitae, rutrum sed dui. Donec vehicula sit amet ex ac finibus. Donec ultrices tellus et laoreet dictum.
See our Picture Gallery!Protocols
Vestibulum nec nisl eu ex ullamcorper mattis ac vel tortor.
Duis nec nibh non nisl tristique condimentum quis eu leo.
Sed venenatis massa in tortor gravida dictum.
Nam sollicitudin enim ac egestas fermentum. Suspendisse tempor urna vel mollis mollis. Proin ac mauris facilisis sapien maximus suscipit nec eget felis. Fusce ac urna sit amet nunc condimentum gravida. Aenean commodo nunc et tempus egestas. Suspendisse cursus quam placerat, vestibulum nunc non, imperdiet felis. Curabitur et erat non justo eleifend commodo. In sit amet sem vitae eros placerat facilisis. Quisque eget ligula vel tellus fermentum vestibulum. Curabitur eu ligula non lorem pulvinar posuere ac commodo ante. Sed convallis quam ut risus dignissim, nec pellentesque risus malesuada. Vestibulum vel sem eu tortor ornare consequat ac eget ligula. Suspendisse eu lacus ut nisi aliquet mollis id nec eros. Integer vulputate sem sed massa porta, eget dapibus odio pellentesque. Morbi sit amet lacus quis urna mattis elementum. See our Lab Notebook!References
[1] Anderson, A. J. and Dawes, E. A. Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiological Reviews 54 (4), 1990. 450 – 472.
[2] Madison, L. L. and Huisman, G. W. Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic. Microbiol Mol Biol Rev 63 (1), 1999. 21 – 53.
[3] Kunasundari, B. and Sudesh, K. Isolation and recovery of microbial polyhydroxyalkanoates. eXPRESS Polymer Letters 5 (7), 2011. 620 – 634.
[4] Leonardi, R. et al. Coenzyme A: Back in action. Progress in Lipid Research 44, 2005. 125 – 153.
[5] Leonardi, R. et al. A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A. The Journal of Biological Chemistry 280, 2005. 3312 – 3322.
[6] Jacquel, N. et al. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering Journal 39 (1), 2008. 15 – 27.
[7] Rahman, A. et al. Secretion of polyhydroxybuterate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering 7 (24), 2013.