Drew Endy

All Publications

• Genome-wide transcription response ofStaphylococcus epidermidisto heat shock and medically relevant glucose levels. Frontiers in microbiology Benjamin, K. N., Goyal, A., Nair, R. V., Endy, D. 2024; 15: 1408796

  Abstract

  Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. We quantified genome-wide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We found 85 of 2,354 genes change up to ~34-fold in response to medically relevant changes in glucose concentration (0-17mM; adj p ≤0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically relevant change in temperature (37-45°C; adj p ≤0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We identified and curated 38 glucose-responsive genes as candidate ON or OFF switches for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms.

  View details for DOI 10.3389/fmicb.2024.1408796

  View details for PubMedID 39104585

• Engineering tRNA abundances for synthetic cellular systems. Nature communications Maheshwari, A. J., Calles, J., Waterton, S. K., Endy, D. 2023; 14 (1): 4594

  Abstract

  Routinizing the engineering of synthetic cells requires specifying beforehand how many of each molecule are needed. Physics-based tools for estimating desired molecular abundances in whole-cell synthetic biology are missing. Here, we use a colloidal dynamics simulator to make predictions for how tRNA abundances impact protein synthesis rates. We use rational design and direct RNA synthesis to make 21 synthetic tRNA surrogates from scratch. We use evolutionary algorithms within a computer aided design framework to engineer translation systems predicted to work faster or slower depending on tRNA abundance differences. We build and test the so-specified synthetic systems and find qualitative agreement between expected and observed systems. First principles modeling combined with bottom-up experiments can help molecular-to-cellular scale synthetic biology realize design-build-work frameworks that transcend tinker-and-test.

  View details for DOI 10.1038/s41467-023-40199-9

  View details for PubMedID 37524714

  View details for PubMedCentralID 307586

• Colloidal Physics Modeling Reveals How Per-Ribosome Productivity Increases with Growth Rate in Escherichia coli. mBio Maheshwari, A. J., Sunol, A. M., Gonzalez, E., Endy, D., Zia, R. N. 2022: e0286522

  Abstract

  Faster-growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes must increase too, almost doubling by an unknown mechanism. Prior models point to diffusive transport as a limiting factor but raise a paradox: faster-growing cells are more crowded, yet crowding slows diffusion. We suspected that physical crowding, transport, and stoichiometry, considered together, might reveal a more nuanced explanation. To investigate, we built a first-principles physics-based model of Escherichia coli cytoplasm in which Brownian motion and diffusion arise directly from physical interactions between individual molecules of finite size, density, and physiological abundance. Using our microscopically detailed model, we predicted that physical transport of individual ternary complexes accounts for ~80% of translation elongation latency. We also found that volumetric crowding increases during faster growth even as cytoplasmic mass density remains relatively constant. Despite slowed diffusion, we predicted that improved proximity between ternary complexes and ribosomes wins out, illustrating a simple physics-based mechanism for how individual elongating ribosomes become more productive. We speculate that crowding imposes a physical limit on growth rate and undergirds cellular behavior more broadly. Unfitted colloidal-scale modeling offers systems biology a complementary "physics engine" for exploring how cellular-scale behaviors arise from physical transport and reactions among individual molecules. IMPORTANCE Ribosomes are the factories in cells that synthesize proteins. When cells grow faster, there are not enough ribosomes to keep up with the demand for faster protein synthesis without individual ribosomes becoming more productive. Yet, faster-growing cells are more crowded, seemingly making it harder for each ribosome to do its work. Our computational model of the physics of translation elongation reveals the underlying mechanism for how individual ribosomes become more productive: proximity and stoichiometry of translation molecules overcome crowding. Our model also suggests a universal physical limitation of cell growth rates.

  View details for DOI 10.1128/mbio.02865-22

  View details for PubMedID 36537810

• Direct and indirect impacts of synthetic biology on biodiversity conservation. iScience Macfarlane, N. B., Adams, J., Bennett, E. L., Brooks, T. M., Delborne, J. A., Eggermont, H., Endy, D., Esvelt, K. M., Kolodziejczyk, B., Kuiken, T., Oliva, M. J., Pena Moreno, S., Slobodian, L., Smith, R. B., Thizy, D., Tompkins, D. M., Wei, W., Redford, K. H. 2022; 25 (11): 105423

  Abstract

  The world's biodiversity is in crisis. Synthetic biology has the potential to transform biodiversity conservation, both directly and indirectly, in ways that are negative and positive. However, applying these biotechnology tools to environmental questions is fraught with uncertainty and could harm cultures, rights, livelihoods, and nature. Decisions about whether or not to use synthetic biology for conservation should be understood alongside the reality of ongoing biodiversity loss. In 2022, the 196 Parties to the United Nations Convention on Biological Diversity are negotiating the post-2020 Global Biodiversity Framework that will guide action by governments and other stakeholders for the next decade to conserve the worlds' biodiversity. To date, synthetic biologists, conservationists, and policy makers have operated in isolation. At this critical time, this review brings these diverse perspectives together and emerges out of the need for a balanced and inclusive examination of the potential application of these technologies to biodiversity conservation.

  View details for DOI 10.1016/j.isci.2022.105423

  View details for PubMedID 36388962

• Modeling the colloidal physics of translation elongation in E. coli Maheshwari, A., Gonzalez-Gonzalez, E., Sunol, A. M., Endy, D., Zia, R. N. CELL PRESS. 2022: 122

  View details for Web of Science ID 000759523000592

• Multicolor plate reader fluorescence calibration. Synthetic biology (Oxford, England) Beal, J., Telmer, C. A., Vignoni, A., Boada, Y., Baldwin, G. S., Hallett, L., Lee, T., Selvarajah, V., Billerbeck, S., Brown, B., Cai, G., Cai, L., Eisenstein, E., Kiga, D., Ross, D., Alperovich, N., Sprent, N., Thompson, J., Young, E. M., Endy, D., Haddock-Angelli, T. 2022; 7 (1): ysac010

  Abstract

  Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

  View details for DOI 10.1093/synbio/ysac010

  View details for PubMedID 35949424

• Correction to 'Fail-safe genetic codes designed to intrinsically contain engineered organisms'. Nucleic acids research Calles, J., Justice, I., Brinkley, D., Garcia, A., Endy, D. 1800

  View details for DOI 10.1093/nar/gkab1285

  View details for PubMedID 34935957

• Growing a circular economy with fungal biotechnology: a white paper. Fungal biology and biotechnology Meyer, V., Basenko, E. Y., Benz, J. P., Braus, G. H., Caddick, M. X., Csukai, M., de Vries, R. P., Endy, D., Frisvad, J. C., Gunde-Cimerman, N., Haarmann, T., Hadar, Y., Hansen, K., Johnson, R. I., Keller, N. P., Krasevec, N., Mortensen, U. H., Perez, R., Ram, A. F., Record, E., Ross, P., Shapaval, V., Steiniger, C., van den Brink, H., van Munster, J., Yarden, O., Wosten, H. A. 2020; 7: 5

  Abstract

  Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation's sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

  View details for DOI 10.1186/s40694-020-00095-z

  View details for PubMedID 32280481

• A View from the Counter Terrorism Foxhole: Drew Endy Counter Terrorism Center at West Point Sentinel (CTC Sentinel) Hummel, S., Cruickshank, P., Rassler, D. 2020; 13 (10): 23-34
• Enabling community-based metrology for wood-degrading fungi. Fungal biology and biotechnology Perez, R., Luccioni, M., Kamakaka, R., Clamons, S., Gaut, N., Stirling, F., Adamala, K. P., Silver, P. A., Endy, D. 2020; 7: 2

  Abstract

  Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings.Results: We show that a widely-available and globally-regularized consumer product (Pringles) can support the growth of wood-degrading fungi, and that growth on Pringles-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to~75%.Conclusions: Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi.

  View details for DOI 10.1186/s40694-020-00092-2

  View details for PubMedID 32206323

• Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell Abbott, T. R., Dhamdhere, G. n., Liu, Y. n., Lin, X. n., Goudy, L. n., Zeng, L. n., Chemparathy, A. n., Chmura, S. n., Heaton, N. S., Debs, R. n., Pande, T. n., Endy, D. n., La Russa, M. F., Lewis, D. B., Qi, L. S. 2020

  Abstract

  The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

  View details for DOI 10.1016/j.cell.2020.04.020

  View details for PubMedID 32353252

• Permutational analysis of Saccharomyces cerevisiae regulatory elements. Synthetic biology (Oxford, England) Dhillon, N., Shelansky, R., Townshend, B., Jain, M., Boeger, H., Endy, D., Kamakaka, R. 2020; 5 (1): ysaa007

  Abstract

  Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5' UTRs and transcription terminators/3' UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.

  View details for DOI 10.1093/synbio/ysaa007

  View details for PubMedID 32775697

• Colloidal hydrodynamics of biological cells: A frontier spanning two fields PHYSICAL REVIEW FLUIDS Maheshwari, A. J., Sunol, A. M., Gonzalez, E., Endy, D., Zia, R. N. 2019; 4 (11)

  View details for DOI 10.1103/PhysRevFluids.4.110506

  View details for Web of Science ID 000496932900006

• Definitive demonstration by synthesis of genome annotation completeness. Proceedings of the National Academy of Sciences of the United States of America Jaschke, P. R., Dotson, G. A., Hung, K. S., Liu, D., Endy, D. 2019

  Abstract

  We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage oX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 Bullavirinae genomes related to oX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with 50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the oX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

  View details for DOI 10.1073/pnas.1905990116

  View details for PubMedID 31719208

• Modeling the Brownian hydrodynamics of intracellular motion Zia, R., Maheshwari, A., Endy, D., Gonzalez, E., Sunol, A. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525055503749

• MILESTONE MEETINGS NATURE Gewin, V., Karim, Q., Endy, D., Taylor, J., Schultz, J. 2019; 571 (7766): S86–S87

  View details for Web of Science ID 000477016700029

• Author Correction: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes. Scientific reports Fujishima, K., Wang, K. M., Palmer, J. A., Abe, N., Nakahigashi, K., Endy, D., Rothschild, L. J. 2019; 9 (1): 6242

  Abstract

  A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

  View details for PubMedID 30976030

• Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes (vol 8, 1776, 2018) SCIENTIFIC REPORTS Fujishima, K., Wang, K. M., Palmer, J. A., Abe, N., Nakahigashi, K., Endy, D., Rothschild, L. J. 2019; 9

  View details for DOI 10.1038/s41598-019-42196-9

  View details for Web of Science ID 000464344100001

• Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic acids research Calles, J. n., Justice, I. n., Brinkley, D. n., Garcia, A. n., Endy, D. n. 2019

  Abstract

  One challenge in engineering organisms is taking responsibility for their behavior over many generations. Spontaneous mutations arising before or during use can impact heterologous genetic functions, disrupt system integration, or change organism phenotype. Here, we propose restructuring the genetic code itself such that point mutations in protein-coding sequences are selected against. Synthetic genetic systems so-encoded should fail more safely in response to most spontaneous mutations. We designed fail-safe codes and simulated their expected effects on the evolution of so-encoded proteins. We predict fail-safe codes supporting expression of 20 or 15 amino acids could slow protein evolution to ∼30% or 0% the rate of standard-encoded proteins, respectively. We also designed quadruplet-codon codes that should ensure all single point mutations in protein-coding sequences are selected against while maintaining expression of 20 or more amino acids. We demonstrate experimentally that a reduced set of 21 tRNAs is capable of expressing a protein encoded by only 20 sense codons, whereas a standard 64-codon encoding is not expressed. Our work suggests that biological systems using rationally depleted but otherwise natural translation systems should evolve more slowly and that such hypoevolvable organisms may be less likely to invade new niches or outcompete native populations.

  View details for DOI 10.1093/nar/gkz745

  View details for PubMedID 31511890

• Tools for Multispecies Futures Journal of Design and Science Haraway, D., Endy, D. 2019; 1 (4)

  View details for DOI 10.21428/7808da6b.05eca6f1

• MILESTONE MEETINGS NATURE Gewin, V., Karim, Q., Endy, D., Taylor, J., Schultz, J. 2018; 564 (7736): S86–S87

  View details for DOI 10.1038/d41586-018-07781-4

  View details for Web of Science ID 000453834900013

  View details for PubMedID 30568220

• Opening options for material transfer. Nature biotechnology Kahl, L., Molloy, J., Patron, N., Matthewman, C., Haseloff, J., Grewal, D., Johnson, R., Endy, D. 2018; 36 (10): 923–27

  View details for PubMedID 30307930

• Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes. Scientific reports Fujishima, K. n., Wang, K. M., Palmer, J. A., Abe, N. n., Nakahigashi, K. n., Endy, D. n., Rothschild, L. J. 2018; 8 (1): 1776

  Abstract

  Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

  View details for PubMedID 29379050

  View details for PubMedCentralID PMC5788988

• Measurements of translation initiation from all 64 codons in E. coli NUCLEIC ACIDS RESEARCH Hecht, A., Glasgow, J., Jaschke, P. R., Bawazer, L. A., Munson, M. S., Cochran, J. R., Endy, D., Salit, M. 2017; 45 (7): 3615-3626

  Abstract

  Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

  View details for DOI 10.1093/nar/gkx070

  View details for Web of Science ID 000399448400011

  View details for PubMedID 28334756

• Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies. ACS synthetic biology Nuñez, I. N., Matute, T. F., Del Valle, I. D., Kan, A., Choksi, A., Endy, D., Haseloff, J., Rudge, T. J., Federici, F. 2017; 6 (2): 256-265

  Abstract

  Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

  View details for DOI 10.1021/acssynbio.6b00149

  View details for PubMedID 27794593

• Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies ACS SYNTHETIC BIOLOGY Nunez, I. N., Matute, T. F., Del Valle, I. D., Kan, A., Choksi, A., Endy, D., Haseloff, J., Rudge, T. J., Federici, F. 2017; 6 (2): 256-265

  Abstract

  Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

  View details for DOI 10.1021/acssynbio.6b00149

  View details for Web of Science ID 000394736400010

• When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins ACS SYNTHETIC BIOLOGY Hecht, A., Endy, D., Salit, M., Munson, M. S. 2016; 5 (9): 1024-1027

  Abstract

  The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins.

  View details for DOI 10.1021/acssynbio.6b00072

  View details for Web of Science ID 000383641400015

  View details for PubMedID 27187075

• Measurement and modeling of intrinsic transcription terminators. Nucleic acids research Cambray, G., Guimaraes, J. C., Mutalik, V. K., Lam, C., Mai, Q. A., Thimmaiah, T., Carothers, J. M., Arkin, A. P., Endy, D. 2016; 44 (14): 7006

  View details for DOI 10.1093/nar/gkw379

  View details for PubMedID 27131373

  View details for PubMedCentralID PMC5001589

• Voices of biotech. Nature biotechnology Amit, I., Baker, D., Barker, R., Berger, B., Bertozzi, C., Bhatia, S., Biffi, A., Demichelis, F., Doudna, J., Dowdy, S. F., Endy, D., Helmstaedter, M., Junca, H., June, C., Kamb, S., Khvorova, A., Kim, D., Kim, J., Krishnan, Y., Lakadamyali, M., Lappalainen, T., Lewin, S., Liao, J., Loman, N., Lundberg, E., Lynd, L., Martin, C., Mellman, I., Miyawaki, A., Mummery, C., Nelson, K., Paz, J., Peralta-Yahya, P., Picotti, P., Polyak, K., Prather, K., Qin, J., Quake, S., Regev, A., Rogers, J. A., Shetty, R., Sommer, M., Stevens, M., Stolovitzky, G., Takahashi, M., Tang, F., Teichmann, S., Torres-Padilla, M., Tripathi, L., Vemula, P., Verdine, G., Vollmer, F., Wang, J., Ying, J. Y., Zhang, F., Zhang, T. 2016; 34 (3): 270-275

  View details for DOI 10.1038/nbt.3502

  View details for PubMedID 26963549

• Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates SCIENCE TRANSLATIONAL MEDICINE Courbet, A., Endy, D., Renard, E., Molina, F., Bonnet, J. 2015; 7 (289)

  Abstract

  Whole-cell biosensors have several advantages for the detection of biological substances and have proven to be useful analytical tools. However, several hurdles have limited whole-cell biosensor application in the clinic, primarily their unreliable operation in complex media and low signal-to-noise ratio. We report that bacterial biosensors with genetically encoded digital amplifying genetic switches can detect clinically relevant biomarkers in human urine and serum. These bactosensors perform signal digitization and amplification, multiplexed signal processing with the use of Boolean logic gates, and data storage. In addition, we provide a framework with which to quantify whole-cell biosensor robustness in clinical samples together with a method for easily reprogramming the sensor module for distinct medical detection agendas. Last, we demonstrate that bactosensors can be used to detect pathological glycosuria in urine from diabetic patients. These next-generation whole-cell biosensors with improved computing and amplification capacity could meet clinical requirements and should enable new approaches for medical diagnosis.

  View details for DOI 10.1126/scitranslmed.aaa3601

  View details for Web of Science ID 000355179800006

  View details for PubMedID 26019219

• The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology NATURE BIOTECHNOLOGY Galdzicki, M., Clancy, K. P., Oberortner, E., Pocock, M., Quinn, J. Y., Rodriguez, C. A., Roehner, N., Wilson, M. L., Adam, L., Anderson, J. C., Bartley, B. A., Beal, J., Chandran, D., Chen, J., Densmore, D., Endy, D., Gruenberg, R., Hallinan, J., Hillson, N. J., Johnson, J. D., Kuchinsky, A., Lux, M., Misirli, G., Peccoud, J., Plahar, H. A., Sirin, E., Stan, G., Villalobos, A., Wipat, A., Gennari, J. H., Myers, C. J., Sauro, H. M. 2014; 32 (6): 545-550

  Abstract

  The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a community-driven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.

  View details for DOI 10.1038/nbt.2891

  View details for PubMedID 24911500

• One-step cloning and chromosomal integration of DNA. ACS synthetic biology St-Pierre, F., Cui, L., Priest, D. G., Endy, D., Dodd, I. B., Shearwin, K. E. 2013; 2 (9): 537-541

  Abstract

  We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.

  View details for DOI 10.1021/sb400021j

  View details for PubMedID 24050148

• Composability of regulatory sequences controlling transcription and translation in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kosuri, S., Goodman, D. B., Cambray, G., Mutalik, V. K., Gao, Y., Arkin, A. P., Endy, D., Church, G. M. 2013; 110 (34): 14024-14029

  Abstract

  The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.

  View details for DOI 10.1073/pnas.1301301110

  View details for Web of Science ID 000323271400076

  View details for PubMedID 23924614

  View details for PubMedCentralID PMC3752251

• Amplifying genetic logic gates. Science Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., Endy, D. 2013; 340 (6132): 599-603

  Abstract

  Organisms must process information encoded via developmental and environmental signals to survive and reproduce. Researchers have also engineered synthetic genetic logic to realize simpler, independent control of biological processes. We developed a three-terminal device architecture, termed the transcriptor, that uses bacteriophage serine integrases to control the flow of RNA polymerase along DNA. Integrase-mediated inversion or deletion of DNA encoding transcription terminators or a promoter modulates transcription rates. We realized permanent amplifying AND, NAND, OR, XOR, NOR, and XNOR gates actuated across common control signal ranges and sequential logic supporting autonomous cell-cell communication of DNA encoding distinct logic-gate states. The single-layer digital logic architecture developed here enables engineering of amplifying logic gates to control transcription rates within and across diverse organisms.

  View details for DOI 10.1126/science.1232758

  View details for PubMedID 23539178

• Measurement and modeling of intrinsic transcription terminators NUCLEIC ACIDS RESEARCH Cambray, G., Guimaraes, J. C., Mutalik, V. K., Lam, C., Quynh-Anh Mai, Q. A., Thimmaiah, T., Carothers, J. M., Arkin, A. P., Endy, D. 2013; 41 (9): 5139-5148

  Abstract

  The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

  View details for DOI 10.1093/nar/gkt163

  View details for Web of Science ID 000318570000040

• Quantitative estimation of activity and quality for collections of functional genetic elements NATURE METHODS Mutalik, V. K., Guimaraes, J. C., Cambray, G., Quynh-Anh Mai, Q. A., Christoffersen, M. J., Martin, L., Yu, A., Lam, C., Rodriguez, C., Bennett, G., Keasling, J. D., Endy, D., Arkin, A. P. 2013; 10 (4): 347-?

  Abstract

  The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts.

  View details for DOI 10.1038/NMETH.2403

  View details for Web of Science ID 000316851100024

  View details for PubMedID 23474467

• Precise and reliable gene expression via standard transcription and translation initiation elements NATURE METHODS Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Quynh-Anh Mai, Q. A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., Endy, D. 2013; 10 (4): 354-?

  Abstract

  An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

  View details for DOI 10.1038/NMETH.2404

  View details for Web of Science ID 000316851100025

  View details for PubMedID 23474465

• Switches, Switches, Every Where, In Any Drop We Drink MOLECULAR CELL Bonnet, J., Endy, D. 2013; 49 (2): 232-233

  Abstract

  In this issue, Broussard et al. (2013) report genetic switches that regulate cell fate selection; a recombinase attachment site is embedded within a repressor coding sequence, such that integration truncates a proteolysis domain, stabilizing the repressor and setting the switch.

  View details for DOI 10.1016/j.molcel.2013.01.005

  View details for Web of Science ID 000314379400005

  View details for PubMedID 23352244

• A survey of enabling technologies in synthetic biology. Journal of biological engineering Kahl, L. J., Endy, D. 2013; 7 (1): 13-?

  Abstract

  Realizing constructive applications of synthetic biology requires continued development of enabling technologies as well as policies and practices to ensure these technologies remain accessible for research. Broadly defined, enabling technologies for synthetic biology include any reagent or method that, alone or in combination with associated technologies, provides the means to generate any new research tool or application. Because applications of synthetic biology likely will embody multiple patented inventions, it will be important to create structures for managing intellectual property rights that best promote continued innovation. Monitoring the enabling technologies of synthetic biology will facilitate the systematic investigation of property rights coupled to these technologies and help shape policies and practices that impact the use, regulation, patenting, and licensing of these technologies.We conducted a survey among a self-identifying community of practitioners engaged in synthetic biology research to obtain their opinions and experiences with technologies that support the engineering of biological systems. Technologies widely used and considered enabling by survey participants included public and private registries of biological parts, standard methods for physical assembly of DNA constructs, genomic databases, software tools for search, alignment, analysis, and editing of DNA sequences, and commercial services for DNA synthesis and sequencing. Standards and methods supporting measurement, functional composition, and data exchange were less widely used though still considered enabling by a subset of survey participants.The set of enabling technologies compiled from this survey provide insight into the many and varied technologies that support innovation in synthetic biology. Many of these technologies are widely accessible for use, either by virtue of being in the public domain or through legal tools such as non-exclusive licensing. Access to some patent protected technologies is less clear and use of these technologies may be subject to restrictions imposed by material transfer agreements or other contract terms. We expect the technologies considered enabling for synthetic biology to change as the field advances. By monitoring the enabling technologies of synthetic biology and addressing the policies and practices that impact their development and use, our hope is that the field will be better able to realize its full potential.

  View details for DOI 10.1186/1754-1611-7-13

  View details for PubMedID 23663447

  View details for PubMedCentralID PMC3684516

• A fully decompressed synthetic bacteriophage empty setX174 genome assembled and archived in yeast VIROLOGY Jaschke, P. R., Lieberman, E. K., Rodriguez, J., Sierra, A., Endy, D. 2012; 434 (2): 278-284

  Abstract

  The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage.

  View details for DOI 10.1016/j.virol.2012.09.020

  View details for Web of Science ID 000312509300018

  View details for PubMedID 23079106

• Refactored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery ACS SYNTHETIC BIOLOGY Ghosh, D., Kohli, A. G., Moser, F., Endy, D., Belcher, A. M. 2012; 1 (12): 576-582

  Abstract

  M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as "refactoring," we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to re-engineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications.

  View details for DOI 10.1021/sb300052u

  View details for Web of Science ID 000312679800002

  View details for PubMedID 23656279

• Rewritable digital data storage in live cells via engineered control of recombination directionality PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bonnet, J., Subsoontorn, P., Endy, D. 2012; 109 (23): 8884-8889

  Abstract

  The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.

  View details for DOI 10.1073/pnas.1202344109

  View details for Web of Science ID 000304991100029

  View details for PubMedID 22615351

  View details for PubMedCentralID PMC3384180

• Engineered cell-cell communication via DNA messaging. Journal of biological engineering Ortiz, M. E., Endy, D. 2012; 6 (1): 16-?

  Abstract

  Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically "regulate transcription."We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis.We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.

  View details for DOI 10.1186/1754-1611-6-16

  View details for PubMedID 22958599

  View details for PubMedCentralID PMC3509006

• Design and analysis of genetically encoded counters 3rd International Winter Conference of the Neural-Network-Society (INNS-WC) / 11th International Conference on Bioinformatics (InCoB) / 3rd International Conference on Computational Systems-Biology and Bioinformatics (CSBio) Subsoontorn, P., Endy, D. ELSEVIER SCIENCE BV. 2012: 43–54

  View details for DOI 10.1016/j.procs.2012.09.006

  View details for Web of Science ID 000313128500005

• Engineered cell-cell communication via DNA messaging JOURNAL OF BIOLOGICAL ENGINEERING Ortiz, M. E., Endy, D. 2012; 6 (1)

  View details for DOI 10.1186/1754-1611-6-16

  View details for Web of Science ID 000208905300016

• Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thomson, T. M., Benjamin, K. R., Bush, A., Love, T., Pincus, D., Resnekov, O., Yu, R. C., Gordon, A., Colman-Lerner, A., Endy, D., Brent, R. 2011; 108 (50): 20265-20270

  Abstract

  Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system. These methods limited variation between independent measurements of protein abundance to a factor of two. We used these measurements together with quantitative models to identify and investigate behaviors of the pheromone response system sensitive to precise abundances. The difference between the maximum and basal signaling output (dynamic range) of the pheromone response MAPK cascade was strongly sensitive to the abundance of Ste5, the MAPK scaffold protein, and absolute system output depended on the amount of Fus3, the MAPK. Additional analysis and experiment suggest that scaffold abundance sets a tradeoff between maximum system output and system dynamic range, a prediction supported by recent experiments.

  View details for DOI 10.1073/pnas.1004042108

  View details for Web of Science ID 000298034800083

  View details for PubMedID 22114196

• Can we grow buildings? Concepts and requirements for automated nano- to meter-scale building ADVANCED ENGINEERING INFORMATICS Rebolj, D., Fischer, M., Endy, D., Moore, T., Sorgo, A. 2011; 25 (2): 390-398

  View details for DOI 10.1016/j.aei.2010.08.006

  View details for Web of Science ID 000290193700023

• Editorial-Synthetic Biology NUCLEIC ACIDS RESEARCH Collins, J. J., Endy, D., Hutchison, C. A., Roberts, R. J. 2010; 38 (8): 2513-2513

  View details for DOI 10.1093/nar/gkq221

  View details for Web of Science ID 000277238900001

  View details for PubMedID 20423911

• Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression PLOS ONE Martin, L., Che, A., Endy, D. 2009; 4 (11)

  Abstract

  The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.Here, we describe the construction and quantitative characterization of Gemini, a fusion between the beta-galactosidase (beta-gal) alpha-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional beta-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of beta-gal activity and decreases the sensitivity of GFP.Gemini is therefore a bifunctional reporter with a wider dynamic range than the beta-gal alpha-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts.

  View details for DOI 10.1371/journal.pone.0007569

  View details for Web of Science ID 000271414600002

  View details for PubMedID 19888458

  View details for PubMedCentralID PMC2766624

• Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of biological engineering Kelly, J. R., Rubin, A. J., Davis, J. H., Ajo-Franklin, C. M., Cumbers, J., Czar, M. J., de Mora, K., Glieberman, A. L., Monie, D. D., Endy, D. 2009; 3: 4-?

  Abstract

  The engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories.We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by approximately 50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards.Relative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.

  View details for DOI 10.1186/1754-1611-3-4

  View details for PubMedID 19298678

  View details for PubMedCentralID PMC2683166

• Determination of cell fate selection during phage lambda infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA St-Pierre, F., Endy, D. 2008; 105 (52): 20705-20710

  Abstract

  Bacteriophage lambda infection of Escherichia coli can result in distinct cell fate outcomes. For example, some cells lyse whereas others survive as lysogens. A quantitative biophysical model of lambda infection supports the hypothesis that spontaneous differences in the timing of individual molecular events during lambda infection leads to variation in the selection of cell fates. Building from this analysis, the lambda lysis-lysogeny decision now serves as a paradigm for how intrinsic molecular noise can influence cellular behavior, drive developmental processes, and produce population heterogeneity. Here, we report experimental evidence that warrants reconsidering this framework. By using cell fractioning, plating, and single-cell fluorescent microscopy, we find that physical differences among cells present before infection bias lambda developmental outcomes. Specifically, variation in cell volume at the time of infection can be used to help predict cell fate: a approximately 2-fold increase in cell volume results in a 4- to 5-fold decrease in the probability of lysogeny. Other cell fate decisions now thought to be stochastic might also be determined by pre-existing variation.

  View details for DOI 10.1073/pnas.0808831105

  View details for Web of Science ID 000262092800027

  View details for PubMedID 19098103

  View details for PubMedCentralID PMC2605630

• The Alpha Project: a model system for systems biology research 1st q-bio Conference on Cellular Information Processing Yu, R. C., Resnekov, O., Abola, A. P., Andrews, S. S., Benjamin, K. R., Bruck, J., Burbulis, I. E., Colman-Lerner, A., Endy, D., Gordon, A., Holl, M., Lok, L., Pesce, C. G., Serra, E., Smith, R. D., Thomson, T. M., Tsong, A. E., Brent, R. INST ENGINEERING TECHNOLOGY-IET. 2008: 222–33

  Abstract

  One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors' experience may provide a productive template for systems biology investigations of other cellular systems.

  View details for DOI 10.1049/iet-syb:20080127

  View details for Web of Science ID 000259928800003

  View details for PubMedID 19045818

• Refinement and standardization of synthetic biological parts and devices NATURE BIOTECHNOLOGY Canton, B., Labno, A., Endy, D. 2008; 26 (7): 787-793

  Abstract

  The ability to quickly and reliably engineer many-component systems from libraries of standard interchangeable parts is one hallmark of modern technologies. Whether the apparent complexity of living systems will permit biological engineers to develop similar capabilities is a pressing research question. We propose to adapt existing frameworks for describing engineered devices to biological objects in order to (i) direct the refinement and use of biological 'parts' and 'devices', (ii) support research on enabling reliable composition of standard biological parts and (iii) facilitate the development of abstraction hierarchies that simplify biological engineering. We use the resulting framework to describe one engineered biological device, a genetically encoded cell-cell communication receiver named BBa_F2620. The description of the receiver is summarized via a 'datasheet' similar to those widely used in engineering. The process of refinement and characterization leading to the BBa_F2620 datasheet may serve as a starting template for producing many standardized genetically encoded objects.

  View details for DOI 10.1038/nbt1413

  View details for Web of Science ID 000257491600023

  View details for PubMedID 18612302

• Genomics - Reconstruction of the Genomes SCIENCE Endy, D. 2008; 319 (5867): 1196-1197

  View details for DOI 10.1126/science.1155749

  View details for Web of Science ID 000253530600024

  View details for PubMedID 18309068

• Stimulus design for model selection and validation in cell signaling PLOS COMPUTATIONAL BIOLOGY Apgar, J. F., Toettcher, J. E., Endy, D., White, F. M., Tidor, B. 2008; 4 (2)

  Abstract

  Mechanism-based chemical kinetic models are increasingly being used to describe biological signaling. Such models serve to encapsulate current understanding of pathways and to enable insight into complex biological processes. One challenge in model development is that, with limited experimental data, multiple models can be consistent with known mechanisms and existing data. Here, we address the problem of model ambiguity by providing a method for designing dynamic stimuli that, in stimulus-response experiments, distinguish among parameterized models with different topologies, i.e., reaction mechanisms, in which only some of the species can be measured. We develop the approach by presenting two formulations of a model-based controller that is used to design the dynamic stimulus. In both formulations, an input signal is designed for each candidate model and parameterization so as to drive the model outputs through a target trajectory. The quality of a model is then assessed by the ability of the corresponding controller, informed by that model, to drive the experimental system. We evaluated our method on models of antibody-ligand binding, mitogen-activated protein kinase (MAPK) phosphorylation and de-phosphorylation, and larger models of the epidermal growth factor receptor (EGFR) pathway. For each of these systems, the controller informed by the correct model is the most successful at designing a stimulus to produce the desired behavior. Using these stimuli we were able to distinguish between models with subtle mechanistic differences or where input and outputs were multiple reactions removed from the model differences. An advantage of this method of model discrimination is that it does not require novel reagents, or altered measurement techniques; the only change to the experiment is the time course of stimulation. Taken together, these results provide a strong basis for using designed input stimuli as a tool for the development of cell signaling models.

  View details for DOI 10.1371/journal.pcbi.0040030

  View details for Web of Science ID 000255407800023

  View details for PubMedID 18282085

• Engineering BioBrick vectors from BioBrick parts. Journal of biological engineering Shetty, R. P., Endy, D., Knight, T. F. 2008; 2: 5-?

  Abstract

  The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts.Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts.We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1) use the process to make and share new BioBrick vectors; (2) expand the current collection of BioBrick vector parts; and (3) characterize and improve the available collection of BioBrick vector parts.

  View details for DOI 10.1186/1754-1611-2-5

  View details for PubMedID 18410688

  View details for PubMedCentralID PMC2373286

• TABASCO: A single molecule, base-pair resolved gene expression simulator BMC BIOINFORMATICS Kosuri, S., Kelly, J. R., Endy, D. 2007; 8

  Abstract

  Experimental studies of gene expression have identified some of the individual molecular components and elementary reactions that comprise and control cellular behavior. Given our current understanding of gene expression, and the goals of biotechnology research, both scientists and engineers would benefit from detailed simulators that can explicitly compute genome-wide expression levels as a function of individual molecular events, including the activities and interactions of molecules on DNA at single base pair resolution. However, for practical reasons including computational tractability, available simulators have not been able to represent genome-scale models of gene expression at this level of detail.Here we develop a simulator, TABASCO http://openwetware.org/wiki/TABASCO, which enables the precise representation of individual molecules and events in gene expression for genome-scale systems. We use a single molecule computational engine to track individual molecules interacting with and along nucleic acid polymers at single base resolution. Tabasco uses logical rules to automatically update and delimit the set of species and reactions that comprise a system during simulation, thereby avoiding the need for a priori specification of all possible combinations of molecules and reaction events. We confirm that single molecule, base-pair resolved simulation using TABASCO (Tabasco) can accurately compute gene expression dynamics and, moving beyond previous simulators, provide for the direct representation of intermolecular events such as polymerase collisions and promoter occlusion. We demonstrate the computational capacity of Tabasco by simulating the entirety of gene expression during bacteriophage T7 infection; for reference, the 39,937 base pair T7 genome encodes 56 genes that are transcribed by two types of RNA polymerases active across 22 promoters.Tabasco enables genome-scale simulation of transcription and translation at individual molecule and single base-pair resolution. By directly representing the position and activity of individual molecules on DNA, Tabasco can directly test the effects of detailed molecular processes on system-wide gene expression. Tabasco would also be useful for studying the complex regulatory mechanisms controlling eukaryotic gene expression. The computational engine underlying Tabasco could also be adapted to represent other types of processive systems in which individual reaction events are organized across a single spatial dimension (e.g., polysaccharide synthesis).

  View details for DOI 10.1186/1471-2105-8-480

  View details for Web of Science ID 000253159400001

  View details for PubMedID 18093293

• Synthetic genomics - Options for governance BIOSECURITY AND BIOTERRORISM-BIODEFENSE STRATEGY PRACTICE AND SCIENCE Garfinkel, M. S., Endy, D., Epstein, G. L., Friedman, R. M. 2007; 5 (4): 359-361

  View details for DOI 10.1089/bsp.2007.0923

  View details for Web of Science ID 000251948300015

  View details for PubMedID 18081496

• DNA synthesis and biological security NATURE BIOTECHNOLOGY Bugl, H., Danner, J. P., Molinari, R. J., Mulligan, J. T., Park, H., Reichert, B., Roth, D. A., Wagner, R., Budowle, B., Scripp, R. M., Smith, J. A., Steele, S. J., Church, G., Endy, D. 2007; 25 (6): 627-629

  View details for DOI 10.1038/nbt0607-627

  View details for Web of Science ID 000247077500014

  View details for PubMedID 17557094

• Engineering life: Building a fab for biology SCIENTIFIC AMERICAN Baker, D., Group, B. I., Church, G., Collins, J., Endy, D., Jacobson, J., Keasling, J., Modrich, P., Smolke, C., Weiss, R. 2006; 294 (6): 44-51

  View details for Web of Science ID 000237445200027

  View details for PubMedID 16711359

• Foundations for engineering biology NATURE Endy, D. 2005; 438 (7067): 449-453

  Abstract

  Engineered biological systems have been used to manipulate information, construct materials, process chemicals, produce energy, provide food, and help maintain or enhance human health and our environment. Unfortunately, our ability to quickly and reliably engineer biological systems that behave as expected remains quite limited. Foundational technologies that make routine the engineering of biology are needed. Vibrant, open research communities and strategic leadership are necessary to ensure that the development and application of biological technologies remains overwhelmingly constructive.

  View details for DOI 10.1038/nature04342

  View details for Web of Science ID 000233458200039

  View details for PubMedID 16306983

• Regulated cell-to-cell variation in a cell-fate decision system NATURE Colman-Lerner, A., Gordon, A., Serra, E., Chin, T., Resnekov, O., Endy, D., Pesce, C. G., Brent, R. 2005; 437 (7059): 699-706

  Abstract

  Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise'). Instead, variation is dominated by differences in the capacity of individual cells to transmit signals through the pathway ('pathway capacity') and to express proteins from genes ('expression capacity'). Cells with high expression capacity express proteins at a higher rate and increase in volume more rapidly. Our results identify two mechanisms that regulate cell-to-cell variation in pathway capacity. First, the MAP kinase Fus3 suppresses variation at high pheromone levels, while the MAP kinase Kss1 enhances variation at low pheromone levels. Second, pathway capacity and expression capacity are negatively correlated, suggesting a compensatory mechanism that allows cells to respond more precisely to pheromone in the presence of a large variation in expression capacity.

  View details for DOI 10.1038/nature03998

  View details for Web of Science ID 000232157900045

  View details for PubMedID 16170311

• Refactoring bacteriophage T7 MOLECULAR SYSTEMS BIOLOGY Chan, L. Y., Kosuri, S., Endy, D. 2005; 1

  Abstract

  Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11,515 base pairs (bp) of the 39,937 bp wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.

  View details for DOI 10.1038/msb4100025

  View details for Web of Science ID 000243244500019

  View details for PubMedID 16729053

  View details for PubMedCentralID PMC1681472

• Signal transduction - Molecular monogamy NATURE Endy, D., Yaffe, M. B. 2003; 426 (6967): 614-615

  View details for DOI 10.1038/426614a

  View details for Web of Science ID 000187132800023

  View details for PubMedID 14668845

• Signal transduction. Decoding NF-kappaB signaling. Science Ting, A. Y., Endy, D. 2002; 298 (5596): 1189-1190

  View details for PubMedID 12424362

• Modelling cellular behaviour NATURE Endy, D., Brent, R. 2001; 409 (6818): 391-395

  View details for Web of Science ID 000166434300061

  View details for PubMedID 11201753

• Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Endy, D., You, L. C., Yin, J., Molineux, I. J. 2000; 97 (10): 5375-5380

  Abstract

  We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 10(5) mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively "nonessential" genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

  View details for Web of Science ID 000086998500068

  View details for PubMedID 10792041

  View details for PubMedCentralID PMC25836

• Toward antiviral strategies that resist viral escape ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Endy, D., Yin, J. 2000; 44 (4): 1097-1099

  Abstract

  We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance.

  View details for Web of Science ID 000085997200049

  View details for PubMedID 10722522

  View details for PubMedCentralID PMC89823

• Intracellular kinetics of a growing virus: A genetically structured simulation for bacteriophage T7 BIOTECHNOLOGY AND BIOENGINEERING Endy, D., Kong, D., Yin, J. 1997; 55 (2): 375-389

  Abstract

  Viruses have evolved to efficiently direct the resources of their hosts toward their own reproduction. A quantitative understanding of viral growth will help researchers develop antiviral strategies, design metabolic pathways, construct vectors for gene therapy, and engineer molecular systems that self-assemble. As a model system we examine here the growth of bacteriophage T7 in Escherichia coli using a chemical-kinetic framework. Data published over the last three decades on the genetics, physiology, and biophysics of phage T7 are incorporated into a genetically structured simulation that accounts for entry of the T7 genome into its host, expression of T7 genes, replication of T7 DNA, assembly of T7 procapsids, and packaging of T7 DNA to finally produce intact T7 progeny. Good agreement is found between the simulated behavior and experimental observations for the shift in transcription capacity from the host to the phage, the initiation times of phage protein synthesis, and the intracellular assembly of both wild-type phage and a fast-growing deletion mutant. The simulation is utilized to predict the effect of antisense molecules targeted to different T7 mRNA. Further, a postulated mechanism for the down regulation of T7 transcription in vivo is quantitatively examined and shown to agree with available data. The simulation is found to be a useful tool for exploring and understanding the dynamics of virus growth at the molecular level. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 375-389, 1997.

  View details for Web of Science ID A1997XH85500015

  View details for PubMedID 18636496