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1. Advanced DNA assembly strategies and standards for synthetic biology

Author

Casini, Arturo, Baldwin, Geoff, and Ellis, Tom

Subjects
570
Abstract

DNA assembly is a fundamental enabling technology for synthetic biology, yet it is also extremely unreliable, expensive and time-consuming. The process usually requires a significant part of total time and effort that can be dedicated to a project, reducing the resources available for the rest of the research, and is also frequently subject to unexpected problems, introducing an undesirable element of unpredictability that might compromise an entire project. This thesis describes the development of three DNA assembly tools that aim to facilitate and speed up synthetic biology research: “MODAL” is a fast and easy to use assembly strategy that brings the advantages of standardisation and modularity to the latest-generation long overlap-based DNA assembly techniques. “Linker” is a software tool that generates DNA sequences specifically optimised to act as high-efficiency homology regions in long overlap-based DNA assembly reactions. Finally with “BASIC” we propose a new DNA assembly standard that incorporates the advances of MODAL and Linker and brings an additional series of improvements in an original assembly workflow. BASIC aims first of all to make DNA assembly significantly more reliable by addressing and/or removing all the unpredictability elements. It also maintains the speed, ease of use and flexibility of MODAL while achieving the same or better efficiency than the best currently available DNA assembly techniques and standards.

Published
2015
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2. A community effort in SARS‐CoV‐2 drug discovery

Author

Schimunek, Johannes, primary, Seidl, Philipp, additional, Elez, Katarina, additional, Hempel, Tim, additional, Le, Tuan, additional, Noé, Frank, additional, Olsson, Simon, additional, Raich, Lluís, additional, Winter, Robin, additional, Gokcan, Hatice, additional, Gusev, Filipp, additional, Gutkin, Evgeny M., additional, Isayev, Olexandr, additional, Kurnikova, Maria G., additional, Narangoda, Chamali H., additional, Zubatyuk, Roman, additional, Bosko, Ivan P., additional, Furs, Konstantin V., additional, Karpenko, Anna D., additional, Kornoushenko, Yury V., additional, Shuldau, Mikita, additional, Yushkevich, Artsemi, additional, Benabderrahmane, Mohammed, additional, Bousquet-Melou, Patrick, additional, Bureau, Ronan, additional, Charton, Beatrice, additional, Cirou, Bertrand, additional, Gil, Gérard, additional, Allen, William J., additional, Sirimulla, Suman, additional, Watowich, Stanley, additional, Antonopoulos, Nick, additional, Epitropakis, Nikolaos, additional, Krasoulis, Agamemnon, additional, Pitsikalis, Vassilis, additional, Theodorakis, Stavros, additional, Kozlovskii, Igor, additional, Maliutin, Anton, additional, Medvedev, Alexander, additional, Popov, Petr, additional, Zaretckii, Mark, additional, Eghbal-zadeh, Hamid, additional, Halmich, Christina, additional, Hochreiter, Sepp, additional, Mayr, Andreas, additional, Ruch, Peter, additional, Widrich, Michael, additional, Berenger, Francois, additional, Kumar, Ashutosh, additional, Yamanishi, Yoshihiro, additional, Zhang, Kam, additional, Bengio, Emmanuel, additional, Bengio, Yoshua, additional, Jain, Moksh, additional, Korablyov, Maksym, additional, Liu, Cheng-Hao, additional, Gilles, Marcous, additional, Glaab, Enrico, additional, Barnsley, Kelly, additional, Iyengar, Suhasini M., additional, Ondrechen, Mary Jo, additional, Haupt, V. Joachim, additional, Kaiser, Florian, additional, Schroeder, Michael, additional, Pugliese, Luisa, additional, Albani, Simone, additional, Athanasiou, Christina, additional, Beccari, Andrea, additional, Carloni, Paolo, additional, D'Arrigo, Giulia, additional, Gianquinto, Eleonora, additional, Goßen, Jonas, additional, Hanke, Anton, additional, Joseph, Benjamin P., additional, Kokh, Daria B., additional, Kovachka, Sandra, additional, Manelfi, Candida, additional, Mukherjee, Goutam, additional, Muñiz-Chicharro, Abraham, additional, Musiani, Francesco, additional, Nunes-Alves, Ariane, additional, Paiardi, Giulia, additional, Rossetti, Giulia, additional, Sadiq, S. Kashif, additional, Spyrakis, Francesca, additional, Talarico, Carmine, additional, Tsengenes, Alexandros, additional, Wade, Rebecca, additional, Copeland, Conner, additional, Gaiser, Jeremiah, additional, Olson, Daniel R., additional, Roy, Amitava, additional, Venkatraman, Vishwesh, additional, Wheeler, Travis J., additional, Arthanari, Haribabu, additional, Blaschitz, Klara, additional, Cespugli, Marco, additional, Durmaz, Vedat, additional, Fackeldey, Konstantin, additional, Fischer, Patrick D., additional, Gorgulla, Christoph, additional, Gruber, Christian, additional, Gruber, Karl, additional, Hetmann, Michael, additional, Kinney, Jamie E., additional, Das, Krishna M. Padmanabha, additional, Pandita, Shreya, additional, Singh, Amit, additional, Steinkellner, Georg, additional, Tesseyre, Guilhem, additional, Wagner, Gerhard, additional, Wang, Zi-Fu, additional, Yust, Ryan J., additional, Druzhilovskiy, Dmitry S., additional, Filimonov, Dmitry, additional, Pogodin, Pavel V., additional, Poroikov, Vladimir, additional, Rudik, Anastassia V., additional, Stolbov, Leonid A., additional, Veselovsky, Alexander V., additional, De Rosa, Maria, additional, Simone, Giada De, additional, Gulotta, Maria R., additional, Lombino, Jessica, additional, Mekni, Nedra, additional, Perricone, Ugo, additional, Casini, Arturo, additional, Embree, Amanda, additional, Gordon, D. Benjamin, additional, Lei, David, additional, Pratt, Katelin, additional, Voigt, Christopher A., additional, Chen, Kuang-Yu, additional, Jacob, Yves, additional, Krischuns, Tim, additional, Lafaye, Pierre, additional, Zettor, Agnès, additional, Rodríguez, M. Luis, additional, White, Kris M., additional, Fearon, Daren, additional, von Delft, Frank, additional, Walsh, Martin A., additional, Horvath, Dragos, additional, Brooks, Charles L., additional, Falsafi, Babak, additional, Ford, Bryan, additional, García-Sastre, Adolfo, additional, Lee, Sang Yup, additional, Naffakh, Nadia, additional, Varnek, Alexandre, additional, Klambauer, Guenter, additional, and Hermans, Thomas M., additional

Published
2023
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3. A community effort to discover small molecule SARS-CoV-2 inhibitors

Author

Schimunek, Johannes, primary, Seidl, Philipp, additional, Elez, Katarina, additional, Hempel, Tim, additional, Le, Tuan, additional, Noé, Frank, additional, Olsson, Simon, additional, Raich, Lluís, additional, Winter, Robin, additional, Gokcan, Hatice, additional, Gusev, Filipp, additional, Gutkin, Evgeny M., additional, Isayev, Olexandr, additional, Kurnikova, Maria G., additional, Narangoda, Chamali H., additional, Zubatyuk, Roman, additional, Bosko, Ivan P., additional, Furs, Konstantin V., additional, Karpenko, Anna D., additional, Kornoushenko, Yury V., additional, Shuldau, Mikita, additional, Yushkevich, Artsemi, additional, Benabderrahmane, Mohammed B., additional, Bousquet-Melou, Patrick, additional, Bureau, Ronan, additional, Charton, Beatrice, additional, Cirou, Bertrand C., additional, Gil, Gérard, additional, Allen, William J., additional, Sirimulla, Suman, additional, Watowich, Stanley, additional, Antonopoulos, Nick A., additional, Epitropakis, Nikolaos E., additional, Krasoulis, Agamemnon K., additional, Pitsikalis, Vassilis P., additional, Theodorakis, Stavros T., additional, Kozlovskii, Igor, additional, Maliutin, Anton, additional, Medvedev, Alexander, additional, Popov, Petr, additional, Zaretckii, Mark, additional, Eghbal-zadeh, Hamid, additional, Halmich, Christina, additional, Hochreiter, Sepp, additional, Mayr, Andreas, additional, Ruch, Peter, additional, Widrich, Michael, additional, Berenger, Francois, additional, Kumar, Ashutosh, additional, Yamanishi, Yoshihiro, additional, Zhang, Kam Y.J., additional, Bengio, Emmanuel, additional, Bengio, Yoshua, additional, Jain, Moksh J., additional, Korablyov, Maksym, additional, Liu, Cheng-Hao, additional, Marcou, Gilles, additional, Glaab, Enrico, additional, Barnsley, Kelly, additional, Iyengar, Suhasini M., additional, Ondrechen, Mary Jo, additional, Haupt, V. Joachim, additional, Kaiser, Florian, additional, Schroeder, Michael, additional, Pugliese, Luisa, additional, Albani, Simone, additional, Athanasiou, Christina, additional, Beccari, Andrea, additional, Carloni, Paolo, additional, D'Arrigo, Giulia, additional, Gianquinto, Eleonora, additional, Goßen, Jonas, additional, Hanke, Anton, additional, Joseph, Benjamin P., additional, Kokh, Daria B., additional, Kovachka, Sandra, additional, Manelfi, Candida, additional, Mukherjee, Goutam, additional, Muñiz-Chicharro, Abraham, additional, Musiani, Francesco, additional, Nunes-Alves, Ariane, additional, Paiardi, Giulia, additional, Rossetti, Giulia, additional, Sadiq, S. Kashif, additional, Spyrakis, Francesca, additional, Talarico, Carmine, additional, Tsengenes, Alexandros, additional, Wade, Rebecca C., additional, Copeland, Conner, additional, Gaiser, Jeremiah, additional, Olson, Daniel R., additional, Roy, Amitava, additional, Venkatraman, Vishwesh, additional, Wheeler, Travis J., additional, Arthanari, Haribabu, additional, Blaschitz, Klara, additional, Cespugli, Marco, additional, Durmaz, Vedat, additional, Fackeldey, Konstantin, additional, Fischer, Patrick D., additional, Gorgulla, Christoph, additional, Gruber, Christian, additional, Gruber, Karl, additional, Hetmann, Michael, additional, Kinney, Jamie E., additional, Padmanabha Das, Krishna M., additional, Pandita, Shreya, additional, Singh, Amit, additional, Steinkellner, Georg, additional, Tesseyre, Guilhem, additional, Wagner, Gerhard, additional, Wang, Zi-Fu, additional, Yust, Ryan J., additional, Druzhilovskiy, Dmitry S., additional, Filimonov, Dmitry A., additional, Pogodin, Pavel V., additional, Poroikov, Vladimir, additional, Rudik, Anastassia V., additional, Stolbov, Leonid A., additional, Veselovsky, Alexander V., additional, De Rosa, Maria, additional, De Simone, Giada, additional, Gulotta, Maria R., additional, Lombino, Jessica, additional, Mekni, Nedra, additional, Perricone, Ugo, additional, Casini, Arturo, additional, Embree, Amanda, additional, Gordon, D. Benjamin, additional, Lei, David, additional, Pratt, Katelin, additional, Voigt, Christopher A., additional, Chen, Kuang-Yu, additional, Jacob, Yves, additional, Krischuns, Tim, additional, Lafaye, Pierre, additional, Zettor, Agnès, additional, Rodríguez, M. Luis, additional, White, Kris M., additional, Fearon, Daren, additional, Von Delft, Frank, additional, Walsh, Martin A., additional, Horvath, Dragos, additional, Brooks III, Charles L., additional, Falsafi, Babak, additional, Ford, Bryan, additional, García-Sastre, Adolfo, additional, Lee, Sang Yup, additional, Naffakh, Nadia, additional, Varnek, Alexandre, additional, Klambauer, Günter, additional, and Hermans, Thomas M., additional

Published
2023
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4. A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology

Author

Casini, Arturo, Chang, Fang-Yuan, Eluere, Raissa, King, Andrew M., Young, Eric M., Dudley, Quentin M., Karim, Ashty, Pratt, Katelin, Bristol, Cassandra, Forget, Anthony, Ghodasara, Amar, Warden-Rothman, Robert, Gan, Rui, Cristofaro, Alexander, Borujeni, Amin Espah, Ryu, Min-Hyung, Li, Jian, Kwon, Yong-Chan, Wang, He, Tatsis, Evangelos, Rodriguez-Lopez, Carlos, O’Connor, Sarah, Medema, Marnix H., Fischbach, Michael A., Jewett, Michael C., Voigt, Christopher, Gordon, D. Benjamin, Casini, Arturo, Chang, Fang-Yuan, Eluere, Raissa, King, Andrew M., Young, Eric M., Dudley, Quentin M., Karim, Ashty, Pratt, Katelin, Bristol, Cassandra, Forget, Anthony, Ghodasara, Amar, Warden-Rothman, Robert, Gan, Rui, Cristofaro, Alexander, Borujeni, Amin Espah, Ryu, Min-Hyung, Li, Jian, Kwon, Yong-Chan, Wang, He, Tatsis, Evangelos, Rodriguez-Lopez, Carlos, O’Connor, Sarah, Medema, Marnix H., Fischbach, Michael A., Jewett, Michael C., Voigt, Christopher, and Gordon, D. Benjamin

Abstract

© 2018 American Chemical Society. Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species (Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Published
2022

6. A pressure test to make 10 molecules in 90 days: External evaluation of methods to engineer biology

Author

Casini, Arturo, Chang, Fang-Yuan, Eluere, Raissa, King, Andrew M, Young, Eric M, Dudley, Quentin M, Karim, Ashty, Pratt, Katelin, Bristol, Cassandra, Forget, Anthony, Ghodasara, Amar, Warden-Rothman, Robert, Gan, Rui, Cristofaro, Alexander, Borujeni, Amin Espah, Ryu, Min-Hyung, Li, Jian, Kwon, Yong-Chan, Wang, He, Tatsis, Evangelos, Rodriguez-Lopez, Carlos, O’Connor, Sarah, Medema, Marnix H, Fischbach, Michael A, Jewett, Michael C, Voigt, Christopher, Gordon, D Benjamin, Casini, Arturo, Chang, Fang-Yuan, Eluere, Raissa, King, Andrew M, Young, Eric M, Dudley, Quentin M, Karim, Ashty, Pratt, Katelin, Bristol, Cassandra, Forget, Anthony, Ghodasara, Amar, Warden-Rothman, Robert, Gan, Rui, Cristofaro, Alexander, Borujeni, Amin Espah, Ryu, Min-Hyung, Li, Jian, Kwon, Yong-Chan, Wang, He, Tatsis, Evangelos, Rodriguez-Lopez, Carlos, O’Connor, Sarah, Medema, Marnix H, Fischbach, Michael A, Jewett, Michael C, Voigt, Christopher, and Gordon, D Benjamin

Abstract

© 2018 American Chemical Society. Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species (Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Published
2021

8. Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG)

Author

Meyer, Adam, primary, Saaem, Ishtiaq, additional, Silverman, Adam, additional, Varaljay, Vanessa A., additional, Mickol, Rebecca, additional, Blum, Steven, additional, Tobias, Alexander V., additional, Schwalm, Nathan D., additional, Mojadedi, Wais, additional, Onderko, Elizabeth, additional, Bristol, Cassandra, additional, Liu, Shangtao, additional, Pratt, Katelin, additional, Casini, Arturo, additional, Eluere, Raissa, additional, Moser, Felix, additional, Drake, Carrie, additional, Gupta, Maneesh, additional, Kelley-Loughnane, Nancy, additional, Lucks, Julius P., additional, Akingbade, Katherine L., additional, Lux, Matthew P., additional, Glaven, Sarah, additional, Crookes-Goodson, Wendy, additional, Jewett, Michael C., additional, Gordon, D. Benjamin, additional, and Voigt, Christopher A., additional

Published
2019
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9. A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology

Author

Casini, Arturo, primary, Chang, Fang-Yuan, additional, Eluere, Raissa, additional, King, Andrew M., additional, Young, Eric M., additional, Dudley, Quentin M., additional, Karim, Ashty, additional, Pratt, Katelin, additional, Bristol, Cassandra, additional, Forget, Anthony, additional, Ghodasara, Amar, additional, Warden-Rothman, Robert, additional, Gan, Rui, additional, Cristofaro, Alexander, additional, Borujeni, Amin Espah, additional, Ryu, Min-Hyung, additional, Li, Jian, additional, Kwon, Yong-Chan, additional, Wang, He, additional, Tatsis, Evangelos, additional, Rodriguez-Lopez, Carlos, additional, O’Connor, Sarah, additional, Medema, Marnix H., additional, Fischbach, Michael A., additional, Jewett, Michael C., additional, Voigt, Christopher, additional, and Gordon, D. Benjamin, additional

Published
2018
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10. A community effort in SARS‐CoV‐2 drug discovery

Author

Schimunek, Johannes, Seidl, Philipp, Elez, Katarina, Hempel, Tim, Le, Tuan, Noé, Frank, Olsson, Simon, Raich, Lluís, Winter, Robin, Gokcan, Hatice, Gusev, Filipp, Gutkin, Evgeny M., Isayev, Olexandr, Kurnikova, Maria G., Narangoda, Chamali H., Zubatyuk, Roman, Bosko, Ivan P., Furs, Konstantin V., Karpenko, Anna D., Kornoushenko, Yury V., Shuldau, Mikita, Yushkevich, Artsemi, Benabderrahmane, Mohammed B., Bousquet‐Melou, Patrick, Bureau, Ronan, Charton, Beatrice, Cirou, Bertrand C., Gil, Gérard, Allen, William J., Sirimulla, Suman, Watowich, Stanley, Antonopoulos, Nick, Epitropakis, Nikolaos, Krasoulis, Agamemnon, Itsikalis, Vassilis, Theodorakis, Stavros, Kozlovskii, Igor, Maliutin, Anton, Medvedev, Alexander, Popov, Petr, Zaretckii, Mark, Eghbal‐Zadeh, Hamid, Halmich, Christina, Hochreiter, Sepp, Mayr, Andreas, Ruch, Peter, Widrich, Michael, Berenger, Francois, Kumar, Ashutosh, Yamanishi, Yoshihiro, Zhang, Kam Y. J., Bengio, Emmanuel, Bengio, Yoshua, Jain, Moksh J., Korablyov, Maksym, Liu, Cheng‐Hao, Marcou, Gilles, Glaab, Enrico, Barnsley, Kelly, Iyengar, Suhasini M., Ondrechen, Mary Jo, Haupt, V. Joachim, Kaiser, Florian, Schroeder, Michael, Pugliese, Luisa, Albani, Simone, Athanasiou, Christina, Beccari, Andrea, Carloni, Paolo, D'Arrigo, Giulia, Gianquinto, Eleonora, Goßen, Jonas, Hanke, Anton, Joseph, Benjamin P., Kokh, Daria B., Kovachka, Sandra, Manelfi, Candida, Mukherjee, Goutam, Muñiz‐Chicharro, Abraham, Musiani, Francesco, Nunes‐Alves, Ariane, Paiardi, Giulia, Rossetti, Giulia, Sadiq, S. Kashif, Spyrakis, Francesca, Talarico, Carmine, Tsengenes, Alexandros, Wade, Rebecca C., Copeland, Conner, Gaiser, Jeremiah, Olson, Daniel R., Roy, Amitava, Venkatraman, Vishwesh, Wheeler, Travis J., Arthanari, Haribabu, Blaschitz, Klara, Cespugli, Marco, Durmaz, Vedat, Fackeldey, Konstantin, Fischer, Patrick D., Gorgulla, Christoph, Gruber, Christian, Gruber, Karl, Hetmann, Michael, Kinney, Jamie E., Padmanabha Das, Krishna M., Pandita, Shreya, Singh, Amit, Steinkellner, Georg, Tesseyre, Guilhem, Wagner, Gerhard, Wang, Zi‐Fu, Yust, Ryan J., Druzhilovskiy, Dmitry S., Filimonov, Dmitry A., Pogodin, Pavel V., Poroikov, Vladimir, Rudik, Anastassia V., Stolbov, Leonid A., Veselovsky, Alexander V., De Rosa, Maria, De Simone, Giada, Gulotta, Maria R., Lombino, Jessica, Mekni, Nedra, Perricone, Ugo, Casini, Arturo, Embree, Amanda, Gordon, D. Benjamin, Lei, David, Pratt, Katelin, Voigt, Christopher A., Chen, Kuang‐Yu, Jacob, Yves, Krischuns, Tim, Lafaye, Pierre, Zettor, Agnès, Rodríguez, M. Luis, White, Kris M., Fearon, Daren, Von Delft, Frank, Walsh, Martin A., Horvath, Dragos, Brooks, Charles L., Falsafi, Babak, Ford, Bryan, García‐Sastre, Adolfo, Yup Lee, Sang, Naffakh, Nadia, Varnek, Alexandre, Klambauer, Günter, and Hermans, Thomas M.

Abstract

The COVID‐19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small‐molecule drugs that are widely available, including in low‐ and middle‐income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the “Billion molecules against COVID‐19 challenge”, to identify small‐molecule inhibitors against SARS‐CoV‐2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find ‘consensus compounds’. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding‐, cleavage‐, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS‐CoV‐2 treatments.

Published
2024
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11. Advanced DNA assembly strategies and standards for synthetic biology

Author

Casini, Arturo, Baldwin, Geoff, Ellis, Tom, and European Commission

Abstract

DNA assembly is a fundamental enabling technology for synthetic biology, yet it is also extremely unreliable, expensive and time-consuming. The process usually requires a significant part of total time and effort that can be dedicated to a project, reducing the resources available for the rest of the research, and is also frequently subject to unexpected problems, introducing an undesirable element of unpredictability that might compromise an entire project. This thesis describes the development of three DNA assembly tools that aim to facilitate and speed up synthetic biology research: “MODAL” is a fast and easy to use assembly strategy that brings the advantages of standardisation and modularity to the latest-generation long overlap-based DNA assembly techniques. “Linker” is a software tool that generates DNA sequences specifically optimised to act as high-efficiency homology regions in long overlap-based DNA assembly reactions. Finally with “BASIC” we propose a new DNA assembly standard that incorporates the advances of MODAL and Linker and brings an additional series of improvements in an original assembly workflow. BASIC aims first of all to make DNA assembly significantly more reliable by addressing and/or removing all the unpredictability elements. It also maintains the speed, ease of use and flexibility of MODAL while achieving the same or better efficiency than the best currently available DNA assembly techniques and standards. Open Access

Published
2014
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16. Four-Step Pathway from Phenylpyruvate to Benzylamine, an Intermediate to the High-Energy Propellant CL-20.

Author

Pandey RP, Casini A, Voigt CA, and Gordon DB

Subjects
Bacterial Proteins genetics, Benzylamines chemistry, Carboxy-Lyases genetics, Escherichia coli chemistry, Escherichia coli genetics, Metabolic Engineering methods, Multigene Family, Oxidoreductases genetics, Phenylpyruvic Acids chemistry, Transaminases genetics, Benzylamines metabolism, Escherichia coli metabolism, Phenylpyruvic Acids metabolism
Abstract

Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli . We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase ( Amycolatopsis orientalis ), a mandelate oxidase ( Streptomyces coelicolor ), a benzoylformate decarboxylase ( Pseudomonas putida ), and an aminotransferase ( Salicibacter pomeroyi ). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of ( S )-mandelic acid, and subsequent genome mining identifies a new mandelate synthase ( Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.

Published
2021
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17. BASIC: A Simple and Accurate Modular DNA Assembly Method.

Author

Storch M, Casini A, Mackrow B, Ellis T, and Baldwin GS

Subjects
Base Sequence, Escherichia coli genetics, Polymerase Chain Reaction, Transformation, Bacterial, Cloning, Molecular
Abstract

Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].

Published
2017
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