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4. The Ketosynthase Domain Controls Chain Length in Mushroom Oligocyclic Polyketide Synthases

Author

Nikolai A. Löhr, Maximilian C. Urban, Frederic Eisen, Lukas Platz, Wolfgang Hüttel, Markus Gressler, Michael Müller, and Dirk Hoffmeister

Subjects
Organic Chemistry, Molecular Medicine, Molecular Biology, Biochemistry
Abstract

The non-reducing iterative type I polyketide synthases (NR-PKSs) CoPKS1 and CoPKS4 of the webcap mushroom Cortinarius odorifer share 88% identical amino acids. CoPKS1 near-exclusively produces a tricyclic octaketide product, atrochrysone carboxylic acid, whereas CoPKS4 shows simultaneous hepta- and octaketide synthase activity and also produces the bicyclic heptaketide 6-hydroxymusizin. To identify the region(s) controlling chain length, four chimeric enzyme variants were constructed and assayed for activity in Aspergillus niger as heterologous expression platform. We provide evidence that the β-ketoacyl synthase (KS) domain determines chain length in these mushroom NR-PKSs, even though their KS domains differ in only ten amino acids. A unique proline-rich linker connecting the acyl carrier protein with the thioesterase domain varies most between these two enzymes but is not involved in chain length control.

Published
2022

6. Publisher Correction: Leveraging implicit knowledge in neural networks for functional dissection and engineering of proteins.

Author

Julius Upmeier zu Belzen, Thore Bürgel, Stefan Holderbach, Felix Bubeck, Lukás Adam, Catharina Gandor, Marita Klein, Jan Mathony, Pauline Pfuderer, Lukas Platz, Moritz Przybilla, Max Schwendemann, Daniel Heid, Mareike Daniela Hoffmann, Michael Jendrusch, Carolin Schmelas, Max Waldhauer, Irina Lehmann, Dominik Niopek, and Roland Eils

Published
2019
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7. Unprecedented Mushroom Polyketide Synthases Produce the Universal Anthraquinone Precursor

Author

Nikolai A. Löhr, Frederic Eisen, Wiebke Thiele, Lukas Platz, Jonas Motter, Wolfgang Hüttel, Markus Gressler, Michael Müller, and Dirk Hoffmeister

Subjects
Polyketides, Anthraquinones, General Chemistry, Agaricales, Polyketide Synthases, Catalysis
Abstract

(Pre-)anthraquinones are widely distributed natural compounds and occur in plants, fungi, microorganisms, and animals, with atrochrysone (1) as the key biosynthetic precursor. Chemical analyses established mushrooms of the genus Cortinarius-the webcaps-as producers of atrochrysone-derived octaketide pigments. However, more recent genomic data did not provide any evidence for known atrochrysone carboxylic acid (4) synthases nor any other polyketide synthase (PKS) producing oligocyclic metabolites. Here, we describe an unprecedented class of non-reducing (NR-)PKS. In vitro assays with recombinant enzyme in combination with in vivo product formation in the heterologous host Aspergillus niger established CoPKS1 and CoPKS4 of C. odorifer as members of a new class of atrochrysone carboxylic acid synthases. CoPKS4 catalyzed both hepta- and octaketide synthesis and yielded 6-hydroxymusizin (6), along with 4. These first mushroom PKSs for oligocyclic products illustrate how the biosynthesis of bioactive natural metabolites evolved independently in various groups of life.

Published
2022

9. Directed evolution of biofuel-responsive biosensors for automated optimization of branched-chain alcohol biosynthesis

Author

Lukas Platz, Gregor W. Schmidt, Maximilian O. Bahls, Sven Panke, and Gaspar Morgado

Subjects
Computer science, High-throughput screening, Bioengineering, Computational biology, Biosensing Techniques, Applied Microbiology and Biotechnology, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Alcohol production, 030304 developmental biology, 0303 health sciences, Ethanol, 030306 microbiology, technology, industry, and agriculture, Branched chain alcohol, Directed evolution, AlkS, Biosensors, Branched-chain higher alcohols (BCHA), chemistry, Metabolic Engineering, Biofuels, Whole cell, Biosensor, Biotechnology
Abstract

The biosynthesis of short-chain alcohols is a carbon-neutral alternative to petroleum-derived production, but strain screening operations are encumbered by laborious analytics. Here, we built, characterized and applied whole cell biosensors by directed evolution of the transcription factor AlkS for screening microbial strain libraries producing industrially relevant alcohols. A selected AlkS variant was applied for in situ product detection in two screening applications concerning key steps in alcohol production. Further, the biosensor strains enabled the implementation of an automated, robotic platform-based workflow with data clustering, which readily allowed the identification of significantly improved strain variants for isopentanol production., Metabolic Engineering, 69, ISSN:1096-7176, ISSN:1096-7184

Published
2022

10. Leveraging implicit knowledge in neural networks for functional dissection and engineering of proteins

Author

Daniel Heid, Dominik Niopek, Max C. Waldhauer, Irina Lehmann, Moritz Jakob Przybilla, Pauline L. Pfuderer, Roland Eils, Thore Bürgel, Catharina Gandor, Marita Klein, Mareike D. Hoffmann, Carolin Schmelas, Felix Bubeck, Julius Upmeier zu Belzen, Lukas Platz, Jan Mathony, Lukas Adam, Stefan Holderbach, Max Schwendemann, and Michael Jendrusch

Subjects
0301 basic medicine, Artificial neural network, Computer Networks and Communications, Computer science, Computational biology, Protein engineering, Molecular machine, Implicit knowledge, Human-Computer Interaction, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Protein sequencing, Signalling, Artificial Intelligence, Leverage (statistics), Computer Vision and Pattern Recognition, Small molecule binding, 030217 neurology & neurosurgery, Software
Abstract

Proteins are nature’s most versatile molecular machines. Deep neural networks trained on large protein datasets have recently been used to tackle the unmet complexity of protein sequence–function relationships. The implicit knowledge contained in these networks represents a powerful, but thus far inaccessible, resource for understanding protein biology. Here, we show that occlusion-based sensitivity analysis can leverage the knowledge present in deep-neural-network-based protein sequence classifiers to identify functionally relevant parts of proteins. We first validated our approach by successfully predicting positions that mediate small molecule binding or catalytic activity across different protein classes. Next, we inferred the impact of point mutations on the activity of ERK and HRas, signalling factors frequently deregulated in cancer. Finally, we used our approach to identify engineering hotspots in CRISPR–Cas9 and anti-CRISPR protein AcrIIA4. Our work demonstrates how implicit knowledge in neural networks can be harnessed for protein functional dissection and protein engineering. Deep neural networks are a powerful tool for predicting protein function, but identifying the specific parts of a protein sequence that are relevant to its functions remains a challenge. An occlusion-based sensitivity technique helps interpret these deep neural networks, and can guide protein engineering by locating functionally relevant protein positions.

Published
2019

11. Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in R. toruloides to include non-ribosomal peptides

Author

Maren Wehrs, Blake A. Simmons, Lukas Platz, Aindrila Mukhopadhyay, Yuzhong Liu, Deepti Tanjore, John M. Gladden, Jadie Moon, Gabriella Papa, Gina M. Geiselman, Eric R. Sundstrom, Todd Pray, Jan Philip Prahl, and Jay D. Keasling

Subjects
Green chemistry, Rhodosporidium toruloides, Heterologous, 010402 general chemistry, 01 natural sciences, Hydrolysate, Pigment, Affordable and Clean Energy, Bioreactor, Environmental Chemistry, SDG 7 - Affordable and Clean Energy, biology, 010405 organic chemistry, Chemistry, Organic Chemistry, Ribosomal RNA, biology.organism_classification, Pollution, Bioproduction, 0104 chemical sciences, Climate Action, Biochemistry, visual_art, Chemical Sciences, visual_art.visual_art_medium, Responsible Consumption and Production
Abstract

Non-ribosomal peptides (NRPs) constitute a diverse class of valuable secondary metabolites, with potential industrial applications including use as pharmaceuticals, polymers and dyes. Current industrial production of dyes is predominantly achieved via chemical synthesis, which can involve toxic precursors and generate hazardous byproducts. Thus, alternative routes of dye production are highly desirable to enhance both workplace safety and environmental sustainability. Correspondingly, biological synthesis of dyes from renewable carbon would serve an ideal green chemistry paradigm. Therefore, we engineered the fungal host Rhodosporidium toruloides to produce the blue pigment indigoidine, an NRP with potential applications in the dye industry, using various low-cost carbon and nitrogen sources. To demonstrate production from renewable carbon sources and assess process scalability we produced indigoidine in 2 L bioreactors, reaching titers of 2.9 ± 0.8 g L-1 using a sorghum lignocellulosic hydrolysate in a batch process and 86.3 ± 7.4 g L-1 using glucose in a high-gravity fed-batch process. This study represents the first heterologous production of an NRP in R. toruloides, thus extending the range of microbial hosts that can be used for sustainable, heterologous production of NRPs. In addition, this is the first demonstration of producing an NRP using lignocellulose. These results highlight the potential of R. toruloides for the sustainable, and scalable production of NRPs, with the highest reported titer of indigoidine or any heterologously produced NRP to date.

Published
2019

12. Publisher Correction: Leveraging implicit knowledge in neural networks for functional dissection and engineering of proteins

Author

Dominik Niopek, Irina Lehmann, Catharina Gandor, Pauline L. Pfuderer, Max Schwendemann, Max C. Waldhauer, Lukas Adam, Moritz Jakob Przybilla, Jan Mathony, Julius Upmeier zu Belzen, Stefan Holderbach, Mareike D. Hoffmann, Lukas Platz, Carolin Schmelas, Felix Bubeck, Marita Klein, Roland Eils, Thore Bürgel, Daniel Heid, and Michael Jendrusch

Subjects
Artificial neural network, Computer Networks and Communications, business.industry, Computer science, Dissection (medical), medicine.disease, Implicit knowledge, Human-Computer Interaction, Artificial Intelligence, medicine, Computer Vision and Pattern Recognition, Artificial intelligence, business, Software
Published
2019

13. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

Author

Jacob, Beal, Trac Haddock Angellii, Markus, Gershater, Kim de Mora, Meagan, Lizarazo, Jim, Hollenhorst, Randy, Rettberg, Philipp, Demling, Rene, Hanke, Michae, Osthegel, Anna, Schechtel, Suresh, Sudarsan, Arne, Zimmermann, Bartosz, Gabryelczyk, Martina, Ikonen, Minnamari, Salmela, Muradıye, Acar, Muhammed Fatih Aktas, Furkan, Bestepe, Furkan Sacit Ceylan, Sadık, Cigdem, Mikail, Dohan, Mustafa, Elitok, Mehmet, Gunduz, Esra, Gunduz, Omer Faruk Hatipoglu, Turan, Kaya, Orhan, Sayin, Safa, Tapan, Osman Faruk Tereci, Abdullah, Uçar, Mustafa, Yilmaz, Jeffrey, Barrick, Alex, Gutierrez, Dennis, Mishler, Jordan, Monk, Kate, Mortensen, Nathan, Shin, Ella, Watkins, Yintong, Chen, Yuji, Jin, Yuanjie, Shi, Haoqian Myelin Zhang, Bruno, Ono, Ieda Maria Martinez Paino, Lais, Ribovski, Ivan, Silva, Danilo Keiji Zampronio, Nils, Birkholz, Rudiger Frederik Busche, Oliver, Konzock, Steffen, Lippold, Carsten, Ludwig, Melanie, Philippi, Lukas, Platz, Christian, Sigismund, Susanne, Weber, Maren, Wehrs, Niels, Werchau, Anna, Wronska, Zen Zen Yen, Yash, Agarwal, Evan, Appleton, Douglas, Densmore, Ariela, Esmurria, Kathleen, Lewis, Alan, Pacheco, Marcel, Bruchez, Danielle, Peters, Cheryl, Telmer, Lena, Wang, Silvia Canas Duarte, Daniel Giraldo Perez, Camilo Gomez Garzon, Jorge Madrid Wolff, Nathaly Marin Medina, Valentina, Mazzanti, Laura Rodriguez Forero, Eitan, Scher, Robin, Dowell, Samantha, O’Hara, Cloe Simone Pogoda, Kendra, Shattuck, Ali, Altintas, Anne Pihl Bali, Rasmus, Bech, Anne, Egholm, Anne Sofie Laerke Hansen, Kristian, Jensen, Kristian Barreth Karlsen, Caroline, Mosbech, Sophia, Belkhelfa, Noemie, Berenger, Romain, Bodinier, Cecile, Jacry, Laura Matabishi Bibi, Pierre, Parutto, Julie, Zaworski, Andries de Vries, Freek de Wijs, Rick, Elbert, Lisa, Hielkema, Chandhuru, Jagadeesan, Bayu, Jayawardhana, Oscar, Kuipers, Anna, Lauxen, Thomas, Meijer, Sandra, Mous, Renske van Raaphorst, Aakanksha, Saraf, Otto, Schepers, Oscar, Smits, Jan Willem Veening, Ruud Detert Oude Weme, Lianne, Wieske, Catherine, Ainsworth, Xenia Spencer Milnes, Alejandro, Gómezávila, Eddie Cano Gamez, Ana Laura Torres Huerta, Carlos Alejandro Meza Ramirez, Philipp, Popp, Jara, Radeck, Anna, Sommer, Xiangkai, Li, Qi, Wu, Hongxia, Zhao, Ruixue, Zhao, Irem, Bastuzel, Yasemin, Ceyhan, Mayda, Gursel, Burak, Kizil, Ilkem, Kumru, Yasemin, Kuvvet, Helin, Tercan, Seniz, Yuksel, Luiza, Niyazmetova, Timothy, Ang, Lucas, Black, Ciaran, Kelly, George, Wadhams, Clovis, Basier, Urszula, Czerwinska, Cindy Suci Ananda, Muhammad Al Azhar, Adelia, Elviantari, Maya, Fitriana, Arief Budi Witarto, Yuliant, Jia Fangxing, Qingfeng, Hou, Wan, Pei, Chen, Rifei, Wang, Rong, Huang, Wei, Zhang, Yushan, Jianguo, He, Dengwen, Lai, Pai, Li, Jianheng, Liu, Chunyang, Ni, Qianbin, Zhang, Cinthya, Cadenas, Zardain Canabal, Eduardo J., Claudia Nallely Alonso Cantu, Mercedes Alejandra Vazquez Cantu, Eduardo Cepeda Canedo, Cesar Miguel Valdez Cordova, Jose Alberto de la Paz Espinosa, Carlos Enrique Alavez Garcia, Ana Laura Navarro Heredia, Adriana, Hernandez, Sebastian Valdivieso Jauregui, Eduardo Ramirez Montiel, Eduardo Serna Morales, Yamile Minerva Castellanos Morales, Omar Alonso Cantu Pena, Ramirez Rodríguez, Eduardo A., Elizabeth Vallejo Trejo, Jesus Gilberto Rodriguez Ceja, Jesus Eduardo Martinez Hernandez, Mario Alberto Pena Hernandez, Enrique Amaya Perez, Rebeca Paola Torres Ramirez, Cla, J., Martin, Hanzel, Sarah Mohand Said, Shihab, Sawar, Dylan, Siriwardena, Alex, Tzahristos, Nils, Anlind, Martin, Friberg, Erik, Gullberg, Stephanie, Herman, Dallin, Christensen, Sara, Gertsch, Cody, Maxfield, Charles, Miller, Ryan, Putman, Christine, Bauerl, Estelles Lopez, Lucia T., Estefania Huet Trujillo, Marta Vazquez Vilar, Marlène Sophie Birk, Nico, Claassens, Walter de Koster, Rik van Rosmalen, Wen Ying Wu, Sian, Davies, Dan, Goss, William, Rostain, Chelsey, Tye, Waqar, Yousaf, Natalie, Farny, Chloe, Lajeunesse, Alex, Turland, Chen, An, Jielin, Chen, Yahong, Chen, Zehua, Che, Baishan, Fang, Xiaotong, Fu, Xifeng, Guo, Yue, Jiang, Yiying, Lei, Jianqiao, Li, Zhe, Li, Chang, Liu, Weibing, Liu, Yang, Li, Yizhu, Lv, Qingyu, Ruan, Yue, Su, Chun, Tang, Yushen, Wang, Fan, Wu, Xiaoshan, Yan, Ruihua, Zhang, Tangduo, Zhang, Farren, Isaacs, Ariel Leyva Hernandez, Natalie, Ma, Stephanie, Mao, Yamini, Naidu, Tuukka, Miinalainen, Marion Aruann, Daniel Calendini, Yoann Chabert, Gael Chambonnier, Myriam, Choukour, Ella de Gaulejac, Camille, Houy, Axel, Levier, Loreen, Logger, Sebastien, Nin, Valerie, Prima, Sturgis, James N., Beibei, Fang, Sadik, Cigdem, Abdullah, Ucar, Alejandro, Gutierrez, Revanth, Poondla, Sanjana, Reddy, Tyler, Rocha, Natalie, Schulte, Devin, Wehle, Marta Eva Jackowski, Sean Ross Craig, Ariana Mirzarafie Ahi, Elliott, Parris, Luba, Prout, Barbara, Steijl, Rachel, Wellman, Zhao, Fan, Zhang, Jing, Yang, Wei, Yang, Yuanzhan, Wen, Zhaosen, Evan, Appletion, Jeffrey, Chen, Abha, Patil, Shaheer, Priracha, Kate, Ryan, Nick, Salvador, John, Viola, Boralli, Camila Maria S., Camila Barbosa Bramorski, Juliana Cancino Bernardi, Ana Laura de Lima, Paula Maria Pincela Lins, Cristiane Casonato Melo, Deborah Cezar Mendonca, Thiago, Mosqueiro, Everton, Silva, Graziele, Vasconcelos, Ruchi, Asthana, Donna, Lee, Michelle, Yu, Peter, Choi, Effie, Lau, Kenneth, Lau, Oscar, Ying, Brandon, Malone, Paul, Young, Aidan, Ceney, Dakota, Hawthorne, Sharon, Lian, Sam, Mellentine, Dylan, Miller, Barbara Castro Moreira, Christie, Peebles, Olivia, Smith, Kevin, Walsh, Allison, Zimont, Michael, Brasino, Michael, Donovan, Hannah, Young, Jan, Bejvl, Daniel, Georgiev, Hynek, Kasl, Katerina Pechotova, Vaclav Pelisek, Anna, Sosnova, Pavel, Zach, Anthony, Ciesla, Benjamin, Hoover, Elliott, Chapman, Jon Marles Wright, Vicky, Moynihan, Liusaidh, Owen, Brooke Rothschild Mancinelli, Emilie, Cuillery, Joseph, Heng, Vincent, Jacquot, Paola, Malsot, Rocco, Meli, Cyril, Pulver, Ari, Sarfatis, Loic, Steiner, Victor, Steininger, Nina van Tiel, Gregoire, Thouvenin, Axel, Uran, Lisa, Baumgartner, Anna, Fomitcheva, Daniel, Gerngross, Verena, Jagger, Michael, Meier, Anja, Michel, Jasmine, Bird, Bradley, Brown, Todd, Burlington, Daniel, Herring, Joseph, Slack, Georgina, Westwood, Emilia, Wojcik, Julian, Bender, Julia, Donauer, Ramona, Emig, Rabea, Jesser, Julika, Neumann, Lara, Stuhn, Takema, Hasegawa, Tomoya, Kozakai, Haruka, Maruyama, Sean, Colloms, Charlotte, Flynn, Vilija, Lomeikaite, James, Provan, Kang, Ning, Shuyan, Tang, Guozhao, Wu, Yunjun, Yang, Zhi, Zeng, Zhan, Yi, Pan, Chu, Jun, Li, Keji, Yan, Athale, Chaitanya A., Swapnil, Bodkhe, Manasi, Gangan, Harsh, Gakhare, Yash, Jawale, Snehal, Kadam, Prachiti, Moghe, Gayatri, Mundhe, Neha, Khetan, Ira, Phadke, Prashant, Uniyal, Siddhesh, Zadey, Ines, Cottignie, Eline, Deprez, Astrid, Deryckere, Jasper, Janssens, Frederik, Jonnaert, Katarzyna, Malczewska, Thomas, Pak, Johan, Robben, Ovia Margaret Thirukkumaran, Vincent Van Deuren, Laurens, Vandebroek, Laura Van Hese, Laetitia Van Wonterghem, Leen, Verschooten, Moritz, Wolter, Joss, Auty, Richard, Badge, Liam, Crawford, Raymond, Dalgleish, Amy, Evans, Cameron, Grundy, Charlie, Kruczko, Payal, Karia, Graeme, Glaister, Rhys, Hakstol, Seme, Mate, Karin, Otero, Dustin, Smith, Jeff, Tingley, Hans Joachim Wieden, Haotian, Wang, Ningning, Yao, Matthias, Franz, Anna, Knoerlein, Nicolas, Koutsoubelis, Loechner, Anne C., Max, Mundt, Alexandra, Richter, Oliver, Schauer, Marjorie, Buss, Sivateja, Tangirala, Brian, Teague, Tianyi, Huang, Xinhao, Song, Yibing, Wei, Zhaoran, Zhang, Longzhi, Cao, Cheng Li, Kang Yang, Zhiqin, Chen, Yuxing, Fang, Libo, Sun, Weiyi, Wang, Yang, Yang, David, Adams, Joshua, Colls, Joshua, Timmons, David, Urick, Julia Anna Adrian, Madina, Akan, Youssef, Chahibi, Rahmi, Lale, Typhaine Le Doujet, Marit Vaagen Roee, Altynay, Abdirakhmanova, Askarbek, Orakov, Azhar, Zhailauova, Jinyang, Liang, Yu, Ma, Qikai, Qin, Yetian, Su, Ju Yeon Han, Raphaella, Hull, Wei Chung Kong, Li Chieh Lu, Duke, Quinton, Pauline, Aubert, Johan, Bourdarias, Olivier, Bugaud, Coralie Demon Chaine, Isabelle, Hatin, Ibtissam Kaid Slimane, Seong Koo Kang, Audrey, Moatti, Cheikh Fall Ndiaye, Mathilde, Ananos, Alexander, Arkhipenko, Valentin, Bailly, Jules, Caput, Javier, Castillo, Alma Chapet Batlle, Floriane, Cherrier, Claudia Demarta Gatsi, Deshmukh, Gopaul, Muriel, Gugger, Caroline, Lambert, Lucas, Krauss, Amelie, Vandendaele, Xiaojing, Li, Lin, Xiaomei, Luo Xunxun, Anders C. h. r. Hansen, Tina, Kronborg, Pettersen, Jens S., Charles, Calvet, Tyler Dae Devlin, Kosuke, Fujishima, Danny, Greenberg, Tina, Ju, Ryan, Kent, Daniel, Kunin, Erica, Lieberman, Griffin, Mccutcheon, Thai, Nguyen, Lynn, Rothschild, Shih, Joseph D., Jack, Takahashi, Kirsten, Thompson, Forrest, Tran, Daniel, Xiang, Felix, Richter, Yang, Xiaoran, Xiangyue, Hu, Changyuan, Deng, Shuyu, Hua, Yumeng, Li, Xinyu, Meng, Boxiang, Wang, Yingqi, Wang, Xuan, Wang, Zixuan, Xu, Jieyu, Yan, Ming, Yan, Yineng, Zhou, Edgar Alberto Alcalá Orozco, José Alberto Cristerna Bermúdez, Daniela Flores Gómez, José Ernesto Hernández Castañeda, Diana Clarisse Montaño Navarro, Juana Yessica PérezÁvila, María Fernanda Salazar Figueroa, María Fernanda Sánchez Arroyo, Oliva Angélica Sánchez Montesinos, Ángel Farid Rojas Cruz, Carlos Ramos Gutiérrez, Alonso Pérez Lona, Carlos Alejandro Meza Ramírez, Fernanda Sotomayor Olivares, Jorge Sebastián Rodríguez Iniesta, Juan Carlos Rueda Silva, Shotaro, Ayukawa, Takahiro, Kashiwagi, Daisuke, Kiga, Misa, Minegishi, Riku, Shinohara, Hiraku, Tokuma, Yuta, Yamazaki, Shuhei, Yasunishi, Erinn Sim Zixuan, Remsha, Afzal, Matthew, Carrigan, Barry, Moran, Marlena, Mucha, Arnas, Petrauskas, Stefan, Marsden, Michelle, Post, Anne, Rodenburg, Hector, Sanguesa, Marit van der Does, Erwin van Rijn, Max van’t Hof, Yeshi de Bruin, Hans de Ferrante, Elles, Elschot, Laura, Jacobs, Jan Willem Muller, Sjoerd, Nooijens, Femke, Vaassen, Cas van der Putten, Esther van Leeuwen, Laura van Smeden, Kwan Kwan Zhu, Kevin, Sabath, Katharina, Sporbeck, Nicolai von Kügelgen, Lisa, Wellinger, Stefanie, Braun, Jack, Ho, Yash, Mishra, Mariola, Sebastian, Lucas von Chamier, Ahsan, Fasih M., Satyadi, Megan A., Vivienne, Gunadhi, Phillip, Kyriakakis, Jenny, Lee, Walter, Thavarajah, Kimia, Abtahi, Robert, Hand, Chun Mun Loke, Adam, Wahab, Iowis, Zhu, Del Bianco, Cristina, Chizzolini, Fabio, Elisa, Godino, Lentini, Roberta, Mansy, Sheref Samir, Yeh Martin, Noel, Claudio Oss Pegorar, Alexander, Cook, Timothy, Kerns, Chad, Nielsen, Michael, Paskett, Alexander, Torgesen, Stephen, Lee, Ophir, Ospovat, Sikandar, Raza, Daniel, Shaykevich, Jarrod, Shilts, Barbora, Bajorinaite, Mykolas, Bendorius, Ieva, Rauluseviciute, Ieva, Savickyte, Sarunas, Tumas, William, Buchser, Elli, Cryan, Caroline, Golino, Andrew, Halleran, Taylor, Jacobs, Michael, Lefew, Joe, Maniaci, John, Marken, Margaret, Saha, Panya, Vij, Kayla, Desanty, Julie, Mazza, Raytheon BBN Technologies, iGEM Foundation, Synthace, Agilent Technology [Santa Clara], Raytheon BBN Technologies, Synthace, and Agilent provided support in the form of salaries for authors JB, MG, and JH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section, The authors wish to thank Sarah Munro and Marc Salit of NIST for help in designing this study. Consortium authors include all persons self-identified by contributing teams as deserving co-authorship credit. Contributors are listed alphabetically within team, and teams alphabetically and by year. Note that some persons may be credited as contributing in both years. Team names are given as identified in iGEM records: full details of each team’s institution and additional members may be found online in the iGEM Foundation archives at:http://year.igem.org/Team:name e.g.: full information on the 2015 ETH_Zurich team may be found at: http://2015.igem.org/Team:ETH_Zurich, BBN Technologies, IGEM Foundation, Synthace Ltd., Agilent Technologies, Department of Biotechnology and Chemical Technology, Helsinki Institute for Information Technology HIIT, ATOMS Turkiye, Boston University, Carnegie Mellon University, Technical University of Denmark, Ludwig Maximilian University of Munich, Middle East Technical University, Sumbawa University of Technology, Southern University of Science and Technology, Sun Yat-Sen University, Tecnológico de Monterrey, Universidad Autonoma de Nuevo Leon, University of Ottawa, Universitat Politècnica de València, Wageningen University and Research Centre, Worcester Polytechnic Institute, Xiamen University, University of Texas at Austin, Bielefeld University, Birkbeck University of London, USP-Brazil, City University of Hong Kong, Colorado State University, CU Boulder, Swiss Federal Institute of Technology Lausanne, Swiss Federal Institute of Technology Zurich, University of Exeter, Indian Institute of Science Education and Research, KU Leuven, Massachusetts Institute of Technology, Northeast Agricultural University, Nanjing Agricultural University, Norwegian University of Science and Technology, Ocean University of China, University of Southern Denmark, Shenzhen Middle School - SZMS 15, Tokyo Institute of Technology, Trinity College Dublin, Delft University of Technology, Eindhoven University of Technology, Tuebingen, University of California Los Angeles, University of California San Diego, University of Maryland, University of Trento, Vanderbilt University, College of William and Mary, Department of Bioproducts and Biosystems, Aalto-yliopisto, Aalto University, Jones, D Dafydd, Discrete Technology and Production Automation, and ​Robotics and image-guided minimally-invasive surgery (ROBOTICS)

Subjects
0106 biological sciences, 0301 basic medicine, green fluorescent protein, Laboratory Proficiency Testing, Transcription, Genetic, International Genetically Engineered Machine, [SDV]Life Sciences [q-bio], lcsh:Medicine, Protein Engineering, 01 natural sciences, Infographics, Synthetic biology, genetics, lcsh:Science, Promoter Regions, Genetic, Macromolecular Engineering, transcription initiation, Measurement, Multidisciplinary, Chemistry, Strain (biology), gene expression regulation, good laboratory practice, Research Assessment, Fluorescence, Reproducibility, 3. Good health, Bioassays and Physiological Analysis, Engineering and Technology, Educational Status, Synthetic Biology, Genetic Engineering, Transcription, Graphs, Research Article, Biotechnology, Transcriptional Activation, Computer and Information Sciences, General Science & Technology, Green Fluorescent Proteins, Bioengineering, Computational biology, iGEM Interlab Study Contributors, Research and Analysis Methods, Promoter Regions, 03 medical and health sciences, promoter region, Genetic, 010608 biotechnology, Escherichia coli, ta215, business.industry, Data Visualization, lcsh:R, Fluorescence Competition, genetic transcription, DNA structure, Reproducibility of Results, Biology and Life Sciences, protein engineering, 030104 developmental biology, Good Health and Well Being, 7 INGENIERÍA Y TECNOLOGÍA, Synthetic Bioengineering, People and Places, lcsh:Q, Population Groupings, biosynthesis, business, metabolism, Undergraduates
Abstract

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. Copyright: © 2016 Beal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Published
2016

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