Browsing by Author "Bennett, Matthew R."
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Item 25th Annual Computational Neuroscience Meeting: CNS-2016(BioMed Central, 2016) Sharpee, Tatyana O.; Destexhe, Alain; Kawato, Mitsuo; Sekulić, Vladislav; Skinner, Frances K.; Wójcik, Daniel K.; Chintaluri, Chaitanya; Cserpán, Dorottya; Somogyvári, Zoltán; Kim, Jae K.; Kilpatrick, Zachary P.; Bennett, Matthew R.; Josić, Kresimir; Elices, Irene; Arroyo, David; Levi, Rafael; Rodriguez, Francisco B.; Varona, Pablo; Hwang, Eunjin; Kim, Bowon; Han, Hio-Been; Kim, Tae; McKenna, James T.; Brown, Ritchie E.; McCarley, Robert W.; Choi, Jee H.; Rankin, James; Popp, Pamela O.; Rinzel, John; Tabas, Alejandro; Rupp, André; Balaguer-Ballester, Emili; Maturana, Matias I.; Grayden, David B.; Cloherty, Shaun L.; Kameneva, Tatiana; Ibbotson, Michael R.; Meffin, Hamish; Koren, Veronika; Lochmann, Timm; Dragoi, Valentin; Obermayer, Klaus; Psarrou, Maria; Schilstra, Maria; Davey, Neil; Torben-Nielsen, Benjamin; Steuber, Volker; Ju, Huiwen; Yu, Jiao; Hines, Michael L.; Chen, Liang; Yu, Yuguo; Kim, Jimin; Leahy, Will; Shlizerman, Eli; Birgiolas, Justas; Gerkin, Richard C.; Crook, Sharon M.; Viriyopase, Atthaphon; Memmesheimer, Raoul-Martin; Gielen, Stan; Dabaghian, Yuri; DeVito, Justin; Perotti, Luca; Kim, Anmo J.; Fenk, Lisa M.; Cheng, Cheng; Maimon, Gaby; Zhao, Chang; Widmer, Yves; Sprecher, Simon; Senn, Walter; Halnes, Geir; Mäki-Marttunen, Tuomo; Keller, Daniel; Pettersen, Klas H.; Andreassen, Ole A.; Einevoll, Gaute T.; Yamada, Yasunori; Steyn-Ross, Moira L.; Alistair Steyn-Ross, D.; Mejias, Jorge F.; Murray, John D.; Kennedy, Henry; Wang, Xiao-Jing; Kruscha, Alexandra; Grewe, Jan; Benda, Jan; Lindner, Benjamin; Badel, Laurent; Ohta, Kazumi; Tsuchimoto, Yoshiko; Kazama, Hokto; Kahng, B.; Tam, Nicoladie D.; Pollonini, Luca; Zouridakis, George; Soh, Jaehyun; Kim, DaeEun; Yoo, Minsu; Palmer, S.E.; Culmone, Viviana; Bojak, Ingo; Ferrario, Andrea; Merrison-Hort, Robert; Borisyuk, Roman; Kim, Chang S.; Tezuka, Taro; Joo, Pangyu; Rho, Young-Ah; Burton, Shawn D.; Bard Ermentrout, G.; Jeong, Jaeseung; Urban, Nathaniel N.; Marsalek, Petr; Kim, Hoon-Hee; Moon, Seok-hyun; Lee, Do-won; Lee, Sung-beom; Lee, Ji-yong; Molkov, Yaroslav I.; Hamade, Khaldoun; Teka, Wondimu; Barnett, William H.; Kim, Taegyo; Markin, Sergey; Rybak, Ilya A.; Forro, Csaba; Dermutz, Harald; Demkó, László; Vörös, János; Babichev, Andrey; Huang, Haiping; Verduzco-Flores, Sergio; Dos Santos, Filipa; Andras, Peter; Metzner, Christoph; Schweikard, Achim; Zurowski, Bartosz; Roach, James P.; Sander, Leonard M.; Zochowski, Michal R.; Skilling, Quinton M.; Ognjanovski, Nicolette; Aton, Sara J.; Zochowski, Michal; Wang, Sheng-Jun; Ouyang, Guang; Guang, Jing; Zhang, Mingsha; Michael Wong, K.Y.; Zhou, Changsong; Robinson, Peter A.; Sanz-Leon, Paula; Drysdale, Peter M.; Fung, Felix; Abeysuriya, Romesh G.; Rennie, Chris J.; Zhao, Xuelong; Choe, Yoonsuck; Yang, Huei-Fang; Mi, Yuanyuan; Lin, Xiaohan; Wu, Si; Liedtke, Joscha; Schottdorf, Manuel; Wolf, Fred; Yamamura, Yoriko; Wickens, Jeffery R.; Rumbell, Timothy; Ramsey, Julia; Reyes, Amy; Draguljić, Danel; Hof, Patrick R.; Luebke, Jennifer; Weaver, Christina M.; He, Hu; Yang, Xu; Ma, Hailin; Xu, Zhiheng; Wang, Yuzhe; Baek, Kwangyeol; Morris, Laurel S.; Kundu, Prantik; Voon, Valerie; Agnes, Everton J.; Vogels, Tim P.; Podlaski, William F.; Giese, Martin; Kuravi, Pradeep; Vogels, Rufin; Seeholzer, Alexander; Podlaski, William; Ranjan, Rajnish; Vogels, Tim; Torres, Joaquin J.; Baroni, Fabiano; Latorre, Roberto; Gips, Bart; Lowet, Eric; Roberts, Mark J.; de Weerd, Peter; Jensen, Ole; van der Eerden, Jan; Goodarzinick, Abdorreza; Niry, Mohammad D.; Valizadeh, Alireza; Pariz, Aref; Parsi, Shervin S.; Warburton, Julia M.; Marucci, Lucia; Tamagnini, Francesco; Brown, Jon; Tsaneva-Atanasova, Krasimira; Kleberg, Florence I.; Triesch, Jochen; Moezzi, Bahar; Iannella, Nicolangelo; Schaworonkow, Natalie; Plogmacher, Lukas; Goldsworthy, Mitchell R.; Hordacre, Brenton; McDonnell, Mark D.; Ridding, Michael C.; Zapotocky, Martin; Smit, Daniel; Fouquet, Coralie; Trembleau, Alain; Dasgupta, Sakyasingha; Nishikawa, Isao; Aihara, Kazuyuki; Toyoizumi, Taro; Robb, Daniel T.; Mellen, Nick; Toporikova, Natalia; Tang, Rongxiang; Tang, Yi-Yuan; Liang, Guangsheng; Kiser, Seth A.; Howard, James H.; Goncharenko, Julia; Voronenko, Sergej O.; Ahamed, Tosif; Stephens, Greg; Yger, Pierre; Lefebvre, Baptiste; Spampinato, Giulia L.B.; Esposito, Elric; et Olivier Marre, Marcel S.; Choi, Hansol; Song, Min-Ho; Chung, SueYeon; Lee, Dan D.; Sompolinsky, Haim; Phillips, Ryan S.; Smith, Jeffrey; Chatzikalymniou, Alexandra P.; Ferguson, Katie; Alex Cayco Gajic, N.; Clopath, Claudia; Angus Silver, R.; Gleeson, Padraig; Marin, Boris; Sadeh, Sadra; Quintana, Adrian; Cantarelli, Matteo; Dura-Bernal, Salvador; Lytton, William W.; Davison, Andrew; Li, Luozheng; Zhang, Wenhao; Wang, Dahui; Song, Youngjo; Park, Sol; Choi, Ilhwan; Shin, Hee-sup; Choi, Hannah; Pasupathy, Anitha; Shea-Brown, Eric; Huh, Dongsung; Sejnowski, Terrence J.; Vogt, Simon M; Kumar, Arvind; Schmidt, Robert; Van Wert, Stephen; Schiff, Steven J.; Veale, Richard; Scheutz, Matthias; Lee, Sang W.; Gallinaro, Júlia; Rotter, Stefan; Rubchinsky, Leonid L.; Cheung, Chung C.; Ratnadurai-Giridharan, Shivakeshavan; Shomali, Safura R.; Ahmadabadi, Majid N.; Shimazaki, Hideaki; Nader Rasuli, S.; Zhao, Xiaochen; Rasch, Malte J.; Wilting, Jens; Priesemann, Viola; Levina, Anna; Rudelt, Lucas; Lizier, Joseph T.; Spinney, Richard E.; Rubinov, Mikail; Wibral, Michael; Bak, Ji H.; Pillow, Jonathan; Zaho, Yuan; Park, Il M.; Kang, Jiyoung; Park, Hae-Jeong; Jang, Jaeson; Paik, Se-Bum; Choi, Woochul; Lee, Changju; Song, Min; Lee, Hyeonsu; Park, Youngjin; Yilmaz, Ergin; Baysal, Veli; Ozer, Mahmut; Saska, Daniel; Nowotny, Thomas; Chan, Ho K.; Diamond, Alan; Herrmann, Christoph S.; Murray, Micah M.; Ionta, Silvio; Hutt, Axel; Lefebvre, Jérémie; Weidel, Philipp; Duarte, Renato; Morrison, Abigail; Lee, Jung H.; Iyer, Ramakrishnan; Mihalas, Stefan; Koch, Christof; Petrovici, Mihai A.; Leng, Luziwei; Breitwieser, Oliver; Stöckel, David; Bytschok, Ilja; Martel, Roman; Bill, Johannes; Schemmel, Johannes; Meier, Karlheinz; Esler, Timothy B.; Burkitt, Anthony N.; Kerr, Robert R.; Tahayori, Bahman; Nolte, Max; Reimann, Michael W.; Muller, Eilif; Markram, Henry; Parziale, Antonio; Senatore, Rosa; Marcelli, Angelo; Skiker, K.; Maouene, M.; Neymotin, Samuel A.; Seidenstein, Alexandra; Lakatos, Peter; Sanger, Terence D.; Menzies, Rosemary J.; McLauchlan, Campbell; van Albada, Sacha J.; Kedziora, David J.; Neymotin, Samuel; Kerr, Cliff C.; Suter, Benjamin A.; Shepherd, Gordon M.G.; Ryu, Juhyoung; Lee, Sang-Hun; Lee, Joonwon; Lee, Hyang J.; Lim, Daeseob; Wang, Jisung; Lee, Heonsoo; Jung, Nam; Anh Quang, Le; Maeng, Seung E.; Lee, Tae H.; Lee, Jae W.; Park, Chang-hyun; Ahn, Sora; Moon, Jangsup; Choi, Yun S.; Kim, Juhee; Jun, Sang B.; Lee, Seungjun; Lee, Hyang W.; Jo, Sumin; Jun, Eunji; Yu, Suin; Goetze, Felix; Lai, Pik-Yin; Kim, Seonghyun; Kwag, Jeehyun; Jang, Hyun J.; Filipović, Marko; Reig, Ramon; Aertsen, Ad; Silberberg, Gilad; Bachmann, Claudia; Buttler, Simone; Jacobs, Heidi; Dillen, Kim; Fink, Gereon R.; Kukolja, Juraj; Kepple, Daniel; Giaffar, Hamza; Rinberg, Dima; Shea, Steven; Koulakov, Alex; Bahuguna, Jyotika; Tetzlaff, Tom; Kotaleski, Jeanette H.; Kunze, Tim; Peterson, Andre; Knösche, Thomas; Kim, Minjung; Kim, Hojeong; Park, Ji S.; Yeon, Ji W.; Kim, Sung-Phil; Kang, Jae-Hwan; Lee, Chungho; Spiegler, Andreas; Petkoski, Spase; Palva, Matias J.; Jirsa, Viktor K.; Saggio, Maria L.; Siep, Silvan F.; Stacey, William C.; Bernar, Christophe; Choung, Oh-hyeon; Jeong, Yong; Lee, Yong-il; Kim, Su H.; Jeong, Mir; Lee, Jeungmin; Kwon, Jaehyung; Kralik, Jerald D.; Jahng, Jaehwan; Hwang, Dong-Uk; Kwon, Jae-Hyung; Park, Sang-Min; Kim, Seongkyun; Kim, Hyoungkyu; Kim, Pyeong S.; Yoon, Sangsup; Lim, Sewoong; Park, Choongseok; Miller, Thomas; Clements, Katie; Ahn, Sungwoo; Ji, Eoon H.; Issa, Fadi A.; Baek, JeongHun; Oba, Shigeyuki; Yoshimoto, Junichiro; Doya, Kenji; Ishii, Shin; Mosqueiro, Thiago S.; Strube-Bloss, Martin F.; Smith, Brian; Huerta, Ramon; Hadrava, Michal; Hlinka, Jaroslav; Bos, Hannah; Helias, Moritz; Welzig, Charles M.; Harper, Zachary J.; Kim, Won S.; Shin, In-Seob; Baek, Hyeon-Man; Han, Seung K.; Richter, René; Vitay, Julien; Beuth, Frederick; Hamker, Fred H.; Toppin, Kelly; Guo, Yixin; Graham, Bruce P.; Kale, Penelope J.; Gollo, Leonardo L.; Stern, Merav; Abbott, L.F.; Fedorov, Leonid A.; Giese, Martin A.; Ardestani, Mohammad H.; Faraji, Mohammad J.; Preuschoff, Kerstin; Gerstner, Wulfram; van Gendt, Margriet J.; Briaire, Jeroen J.; Kalkman, Randy K.; Frijns, Johan H.M.; Lee, Won H.; Frangou, Sophia; Fulcher, Ben D.; Tran, Patricia H.P.; Fornito, Alex; Gliske, Stephen V.; Lim, Eugene; Holman, Katherine A.; Fink, Christian G.; Kim, Jinseop S.; Mu, Shang; Briggman, Kevin L.; Sebastian Seung, H.; Wegener, Detlef; Bohnenkamp, Lisa; Ernst, Udo A.; Devor, Anna; Dale, Anders M.; Lines, Glenn T.; Edwards, Andy; Tveito, Aslak; Hagen, Espen; Senk, Johanna; Diesmann, Markus; Schmidt, Maximilian; Bakker, Rembrandt; Shen, Kelly; Bezgin, Gleb; Hilgetag, Claus-Christian; van Albada, Sacha J.; Sun, Haoqi; Sourina, Olga; Huang, Guang-Bin; Klanner, Felix; Denk, Cornelia; Glomb, Katharina; Ponce-Alvarez, Adrián; Gilson, Matthieu; Ritter, Petra; Deco, Gustavo; Witek, Maria A.G.; Clarke, Eric F.; Hansen, Mads; Wallentin, Mikkel; Kringelbach, Morten L.; Vuust, Peter; Klingbeil, Guido; De Schutter, Erik; Chen, Weiliang; Zang, Yunliang; Hong, Sungho; Takashima, Akira; Zamora, Criseida; Gallimore, Andrew R.; Goldschmidt, Dennis; Manoonpong, Poramate; Karoly, Philippa J.; Freestone, Dean R.; Soundry, Daniel; Kuhlmann, Levin; Paninski, Liam; Cook, Mark; Lee, Jaejin; Fishman, Yonatan I.; Cohen, Yale E.; Roberts, James A.; Cocchi, Luca; Sweeney, Yann; Lee, Soohyun; Jung, Woo-Sung; Kim, Youngsoo; Jung, Younginha; Song, Yoon-Kyu; Chavane, Frédéric; Soman, Karthik; Muralidharan, Vignesh; Srinivasa Chakravarthy, V.; Shivkumar, Sabyasachi; Mandali, Alekhya; Pragathi Priyadharsini, B.; Mehta, Hima; Davey, Catherine E.; Brinkman, Braden A.W.; Kekona, Tyler; Rieke, Fred; Buice, Michael; De Pittà, Maurizio; Berry, Hugues; Brunel, Nicolas; Breakspear, Michael; Marsat, Gary; Drew, Jordan; Chapman, Phillip D.; Daly, Kevin C.; Bradle, Samual P.; Seo, Sat B.; Su, Jianzhong; Kavalali, Ege T.; Blackwell, Justin; Shiau, LieJune; Buhry, Laure; Basnayake, Kanishka; Lee, Sue-Hyun; Levy, Brandon A.; Baker, Chris I; Leleu, Timothée; Philips, Ryan T.; Chhabria, KarishmaItem Embargo A Bacterial Toolkit for Rapid Prototyping of Multicistronic Genetic Circuits from Interchangeable Parts(2024-05-17) Bhakta, Shyam Pravin; Bennett, Matthew R.Bacteria are the most popular chassis for synthetic biology and industrial biotechnology, owing to the comparative ease of their genetic engineering toward desired functions. Synthetic biology has enabled this goal through systematic gene expression control, by abstracting gene regulatory elements as plug and play modules, including promoters, ribosome binding sites, protein-coding sequences, and terminators. These parts are not perfectly modular and frequently fail to function as predicted in a gene or circuit, owing to compositional effects, chassis environment, etc. Furthermore, the specific parts needed to meet a circuit’s performance criteria are often unknown. Rapid prototyping of DNA is necessary to eliminate bottlenecks in the design-build-test cycle of fast-growing bacteria, and physical standardization and sharing of genetic parts and modules across research labs is a promising solution. The Bacterial Toolkit hierarchical DNA assembly system developed in this thesis and the companion part collection allows complete specification of transcriptional and translational cassette elements in custom combinations of mono- and polycistronic operons in any orientation, to efficiently build bacterial genetic circuits, pathways, and vectors for diverse needs from physically interchangeable parts. A distribution of modern gene expression parts is also provided, including promoters, insulators, insulated ribosome binding sites, transcription factors, protein tags, strong terminators, common E. coli and broad-host range origins, selection markers, plasmid maintenance factors, and E. coli genomic integration homologies. The Bacterial Toolkit is already in use by researchers and has been used for the construction of numerous genetic circuits with functions spanning bacterial pattern formation and population control, asymmetric plasmid partitioning, RNA logic, gene expression library construction, glucose biosensing, bioremediation, protein biomaterials, and environmental monitoring of horizontal gene transfer. Here, we used the Toolkit to demonstrate high assembly fidelity and streamlined genomic integration, test transcriptional insulation afforded by connector elements, investigate the design strategies for UTR insulation in operons, and measure the effects of gene compositional contexts on promoter performance.Item A synthetic system for asymmetric cell division in Escherichia coli(2019-07-16) Molinari, Sara; Bennett, Matthew R.; Wagner, Daniel S.One defining property of stem cells is their ability to differentiate via asymmetric cell division, in which a stem cell creates a differentiated daughter cell but retains its own phenotype. Here, I describe a synthetic genetic circuit for controlling asymmetric cell division in E. coli in which a progenitor cell creates a differentiated daughter cell while retaining its original phenotype. Specifically, an inducible system was engineered that can bind and segregate plasmid DNA to a single position in the cell. Upon division, the colocalized plasmids are kept by one and only one of the daughter cells. The other daughter cell receives no plasmid DNA and is hence irreversibly differentiated from its sibling. In this way, asymmetric cell division happens though asymmetric plasmid partitioning. This system was further used to achieve physical separation of genetically distinct cells by tying motility to differentiation. Finally, an orthogonal inducible circuit was characterized that enables the simultaneous asymmetric partitioning of two plasmid species, resulting in pluripotent cells that have four distinct differentiated states. These results point the way towards engineering multicellular systems from prokaryotic hosts.Item AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators(Elsevier, 2016) Sousa, Filipa L.; Parente, Daniel J.; Shis, David L.; Hessman, Jacob A.; Chazelle, Allen; Bennett, Matthew R.; Teichmann, Sarah A.; Swint-Kruse, Liskin; Bioengineering; BiosciencesProtein families evolve functional variation by accumulating point mutations at functionally important amino acid positions. Homologs in the LacI/GalR family of transcription regulators have evolved to bind diverse DNA sequences and allosteric regulatory molecules. In addition to playing key roles in bacterial metabolism, these proteins have been widely used as a model family for benchmarking structural and functional prediction algorithms. We have collected manually curated sequence alignments for >ﾠ3000 sequences, in vivo phenotypic and biochemical data for >ﾠ5750 LacI/GalR mutational variants, and noncovalent residue contact networks for 65 LacI/GalR homolog structures. Using this rich data resource, we compared the noncovalent residue contact networks of the LacI/GalR subfamilies to design and experimentally validate an allosteric mutant of a synthetic LacI/GalR repressor for use in biotechnology. The AlloRep database (freely available at www.AlloRep.org) is a key resource for future evolutionary studies of LacI/GalR homologs and for benchmarking computational predictions of functional change.Item An all-optical biological function generator and oscilloscope framework for characterizing gene circuit dynamics(2014-04-21) Olson, Evan James; Tabor, Jeffrey J.; Igoshin, Oleg A.; Bennett, Matthew R.Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic ‘function generator’ for programming tailor-made gene expression signals in live E. coli. We designed light sequences with experimentally calibrated models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time-courses. This approach enabled generation of accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamic analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.Item Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication(American Chemical Society, 2013) Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.; Bioengineering; Biosciences; Chemical and Biomolecular Engineering; Earth, Environmental and Planetary SciencesCharcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell–cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.Item Bistability and oscillations in co-repressive synthetic microbial consortia(Springer, 2017) Sadeghpour, Mehdi; Veliz-Cuba, Alan; Orosz, Gábor; Josić, Krešimir; Bennett, Matthew R.; Bioengineering; BiosciencesBackground: Synthetic microbial consortia are conglomerations of genetically engineered microbes programmed to cooperatively bring about population-level phenotypes. By coordinating their activity, the constituent strains can display emergent behaviors that are difficult to engineer into isogenic populations. To do so, strains are engineered to communicate with one another through intercellular signaling pathways that depend on cell density. Methods: Here, we used computational modeling to examine how the behavior of synthetic microbial consortia results from the interplay between population dynamics governed by cell growth and internal transcriptional dynamics governed by cell-cell signaling. Specifically, we examined a synthetic microbial consortium in which two strains each produce signals that down-regulate transcription in the other. Within a single strain this regulatory topology is called a “co-repressive toggle switch” and can lead to bistability. Results: We found that in co-repressive synthetic microbial consortia the existence and stability of different states depend on population-level dynamics. As the two strains passively compete for space within the colony, their relative fractions fluctuate and thus alter the strengths of intercellular signals. These fluctuations drive the consortium to alternative equilibria. Additionally, if the growth rates of the strains depend on their transcriptional states, an additional feedback loop is created that can generate oscillations. Conclusions: Our findings demonstrate that the dynamics of microbial consortia cannot be predicted from their regulatory topologies alone, but are also determined by interactions between the strains. Therefore, when designing synthetic microbial consortia that use intercellular signaling, one must account for growth variations caused by the production of protein.Item Emergent genetic oscillations in a synthetic microbial consortium(American Association for the Advancement of Science, 2015) Chen, Ye; Kim, Jae Kyoung; Hirning, Andrew J.; Josić, Krešimir; Bennett, Matthew R.; Institute of Biosciences and BioengineeringA challenge of synthetic biology is the creation of cooperative microbial systems that exhibit population-level behaviors. Such systems use cellular signaling mechanisms to regulate gene expression across multiple cell types. We describe the construction of a synthetic microbial consortium consisting of two distinct cell types—an "activator" strain and a "repressor" strain. These strains produced two orthogonal cell-signaling molecules that regulate gene expression within a synthetic circuit spanning both strains. The two strains generated emergent, population-level oscillations only when cultured together. Certain network topologies of the two-strain circuit were better at maintaining robust oscillations than others. The ability to program population-level dynamics through the genetic engineering of multiple cooperative strains points the way toward engineering complex synthetic tissues and organs with multiple cell types.Item Emergent spatiotemporal population dynamics with cell-length control of synthetic microbial consortia(Public Library of Science, 2021) Winkle, James J.; Karamched, Bhargav R.; Bennett, Matthew R.; Ott, William; Josić, Krešimir; Bioengineering; BiosciencesThe increased complexity of synthetic microbial biocircuits highlights the need for distributed cell functionality due to concomitant increases in metabolic and regulatory burdens imposed on single-strain topologies. Distributed systems, however, introduce additional challenges since consortium composition and spatiotemporal dynamics of constituent strains must be robustly controlled to achieve desired circuit behaviors. Here, we address these challenges with a modeling-based investigation of emergent spatiotemporal population dynamics using cell-length control in monolayer, two-strain bacterial consortia. We demonstrate that with dynamic control of a strain’s division length, nematic cell alignment in close-packed monolayers can be destabilized. We find that this destabilization confers an emergent, competitive advantage to smaller-length strains—but by mechanisms that differ depending on the spatial patterns of the population. We used complementary modeling approaches to elucidate underlying mechanisms: an agent-based model to simulate detailed mechanical and signaling interactions between the competing strains, and a reductive, stochastic lattice model to represent cell-cell interactions with a single rotational parameter. Our modeling suggests that spatial strain-fraction oscillations can be generated when cell-length control is coupled to quorum-sensing signaling in negative feedback topologies. Our research employs novel methods of population control and points the way to programming strain fraction dynamics in consortial synthetic biology.Item Evolutionary Trajectories to Daptomycin Resistance in Enterococcus faecalis(2013-11-18) Miller, Corwin; Olson, John S.; Shamoo, Yousif; Bennett, Matthew R.; Rudolf, Volker H. W.; Tao, Yizhi JaneWith increasing amounts of hospital-acquired antibiotic resistant infections each year and staggering healthcare costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. In this thesis, I have combined experimental evolution, comparative whole genome sequencing, and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin resistant clinical pathogen Enterococcus faecalis strain S613. Maintaining cells inside a turbidostat, a single polymorphic culture was grown sustaining both planktonic and non-planktonic (e.g. biofilm) populations in co-culture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. This approach revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Genetic and phenotypic comparisons between resistant isolates also identified convergent evolutionary trajectories, suggesting a common biochemical mechanism of resistance. Despite the relative ecological simplicity of this approach compared to the complexity of the human body, I show that experimental evolution can be used to rapidly identify clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.Item Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway(Springer Nature, 2021) Ho, Joanne M.L.; Miller, Corwin A.; Smith, Kathryn A.; Mattia, Jacob R.; Bennett, Matthew R.; Bioengineering; BiosciencesPyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.Item Indirect Enrichment of Desirable, but Less Fit Phenotypes, from a Synthetic Microbial Community Using Microdroplet Confinement(American Chemical Society, 2023) Prabhakar, Ramya Ganiga; Fan, Gaoyang; Alnahhas, Razan N.; Hirning, Andrew J.; Bennett, Matthew R.; Shamoo, Yousif; Bioengineering; BiosciencesSpatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was composed of three strains: a “Producer” that makes the diffusible quorum sensing molecule (N-(3-oxododecanoyl)-l-homoserine lactone, C12-oxo-HSL) or AHL; a “Receiver” that is killed by AHL; and a Non-Producer or “cheater” that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allows a more efficient but transient enrichment of more rare and slower-growing Producer subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.Item Majority sensing in synthetic microbial consortia(Springer Nature, 2020) Alnahhas, Razan N.; Sadeghpour, Mehdi; Chen, Ye; Frey, Alexis A.; Ott, William; Josić, Krešimir; Bennett, Matthew R.; Bioengineering; BiosciencesAs synthetic biocircuits become more complex, distributing computations within multi-strain microbial consortia becomes increasingly beneficial. However, designing distributed circuits that respond predictably to variation in consortium composition remains a challenge. Here we develop a two-strain gene circuit that senses and responds to which strain is in the majority. This involves a co-repressive system in which each strain produces a signaling molecule that signals the other strain to down-regulate production of its own, orthogonal signaling molecule. This co-repressive consortium links gene expression to ratio of the strains rather than population size. Further, we control the cross-over point for majority via external induction. We elucidate the mechanisms driving these dynamics by developing a mathematical model that captures consortia response as strain fractions and external induction are varied. These results show that simple gene circuits can be used within multicellular synthetic systems to sense and respond to the state of the population.Item Measuring Competitive Fitness in Dynamic Environments(American Chemical Society, 2013) Razinkov, Ivan A.; Baumgartner, Bridget L.; Bennett, Matthew R.; Tsimring, Lev S.; Hasty, Jeff; Institute of Biosciences and BioengineeringMost yeast genes are dispensable for optimal growth in laboratory cultures. However, this apparent lack of fitness contribution is difficult to reconcile with the theory of natural selection. Here we use stochastic modeling to show that environmental fluctuations can select for a genetic mechanism that does not affect growth in static laboratory environments. We then present a novel experimental platform for measuring the fitness levels of specific genotypes in fluctuating environments. We test this platform by monitoring a mixed culture of two yeast strains that differ in their ability to respond to changes in carbon source yet exhibit the same fitness level in static conditions. When the sugar in the growth medium was switched between galactose and glucose, the wild-type strain gained a growth advantage over the mutant strain. Interestingly, both our computational and experimental results show that the strength of the adaptive advantage conveyed by the wild-type genotype depends on the total number of carbon source switches, not on the frequency of these fluctuations. Our results illustrate the selective power of environmental fluctuations on seemingly slight phenotypic differences in cellular response dynamics and underscore the importance of dynamic processes in the evolution of species.Item Modeling delay in genetic networks: From delay birth-death processes to delay stochastic differential equations(AIP Publishing, 2014) Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Bennett, Matthew R.; Josić, Krešimir; Ott, William; Institute of Biosciences and BioengineeringDelay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay.Item Modeling mechanical interactions in growing populations of rod-shaped bacteria(IOP Publishing, 2017) Winkle, James J.; Igoshin, Oleg A.; Bennett, Matthew R.; Josić, Krešimir; Ott, William; Bioengineering; BiosciencesAdvances in synthetic biology allow us to engineer bacterial collectives with pre-specified characteristics. However, the behavior of these collectives is difficult to understand, as cellular growth and division as well as extra-cellular fluid flow lead to complex, changing arrangements of cells within the population. To rationally engineer and control the behavior of cell collectives we need theoretical and computational tools to understand their emergent spatiotemporal dynamics. Here, we present an agent-based model that allows growing cells to detect and respond to mechanical interactions. Crucially, our model couples the dynamics of cell growth to the cell's environment: Mechanical constraints can affect cellular growth rate and a cell may alter its behavior in response to these constraints. This coupling links the mechanical forces that influence cell growth and emergent behaviors in cell assemblies. We illustrate our approach by showing how mechanical interactions can impact the dynamics of bacterial collectives growing in microfluidic traps.Item Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras(American Chemical Society, 2014) Shis, David L.; Hussain, Faiza; Meinhardt, Sarah; Swint-Kruse, Liskin; Bennett, Matthew R.In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.Item Peroxisome Biogenesis in Drosophila melanogaster: Protein Trafficking, Lipid Metabolism, and Muscle Function(2013-12-02) Faust, Joseph; McNew, James A.; Bartel, Bonnie; Diehl, Michael R.; Stern, Michael; Bennett, Matthew R.Peroxisomes are ubiquitous organelles required for many essential functions, such as fatty acid metabolism. Defects in peroxisome biogenesis cause a spectrum of human diseases known as peroxisome biogenesis disorders (PBDs). These devastating diseases lack effective therapies and it is unclear how peroxisome dysfunction causes the disease state. Animal models are needed to understand the connection between peroxisome biology and animal physiology. The fruit fly, Drosophila melanogaster, has recently become an important animal model in the study of peroxisomes. We have identified the major peroxisomal proteins and pathways in flies and examined peroxisomal protein trafficking. We have found that fruit fly peroxisomes share many features in common with higher animals, but display some important differences. Flies appear to have lost one of the pathways used in other organisms to target proteins to the peroxisomal matrix. Also some proteins are dually localized to peroxisomes and the cytoplasm likely through a weak interaction with the protein machinery that brings peroxisomal proteins into the organelle. We have also generated fly mutants with impaired peroxisome biogenesis and shown that peroxisomes are required for normal development and lipid metabolism. Flies with impaired peroxisome biogenesis also show defects in multiple processes that depend on muscle function, such as locomotion. PBD patients also display muscle defects, but it is thought to be a secondary effect of neuronal dysfunction. We propose that peroxisome loss in humans, like in flies, may directly affect muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in fly physiology and specifically in muscle cells may reveal novel aspects of PBD etiology.Item A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria(Springer Nature, 2023) Liu, Baiyang; Samaniego, Christian Cuba; Bennett, Matthew R.; Franco, Elisa; Chappell, James; Bioengineering; BiosciencesA lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.Item Sources of Variability in a Synthetic Gene Oscillator(Public Library of Science, 2015) Veliz-Cuba, Alan; Hirning, Andrew J.; Atanas, Adam A.; Hussain, Faiza; Vancia, Flavia; Josić, Krešimir; Bennett, Matthew R.; Bioengineering; BiosciencesSynthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits.