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  • Rational Design of an Anticalin-Type Sugar-Binding Protein Using a Genetically Encoded Boronate Side Chain
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-22
    Selvakumar Edwardraja, Andreas Eichinger, Ina Theobald, Carina Andrea Sommer, Andreas J. Reichert, Arne Skerra

    The molecular recognition of carbohydrates plays a fundamental role in many biological processes. However, the development of carbohydrate-binding reagents for biomedical research and use poses a challenge due to the generally poor affinity of proteins towards sugars in aqueous solution. Here, we describe the effective molecular recognition of pyranose monosaccharides (in particular, galactose and mannose) by a rationally designed protein receptor based on the human lipocalin scaffold (Anticalin). Complexation relies on reversible covalent cis-diol boronate diester formation with a genetically encoded L-boronophenylalanine (Bpa) residue which was incorporated as a non-natural amino acid at a sterically permissive position in the binding site of the Anticalin, as confirmed by X-ray crystallography. Com-pared with the metal-ion and/or avidity-dependent oligovalent lectins that prevail in nature, our approach offers a novel and promising route to generate tight sugar-binding reagents both as research reagents and for biomedical applications.

    更新日期:2017-09-23
  • Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-21
    S. M. Nayeemul Bari, Forrest C. Walker, Katie Cater, Barbaros Aslan, Asma Hatoum-Aslan
    更新日期:2017-09-21
  • Growing membranes in vitro by continuous phospholipid biosynthesis from free fatty acids
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-18
    Marten Exterkate, Antonella Caforio, Marc C. A. Stuart, Arnold J.M. Driessen

    One of the key aspects that defines a cell as a living entity is its ability to self-reproduce. In this process, membrane biogenesis is an essential element. Here, we developed an in vitro phospholipid biosynthesis pathway based on a cascade of eight enzymes, starting from simple fatty acid building blocks and glycerol 3-phosphate. The reconstituted system yields multiple phospholipid species that vary in acyl-chain and polar head group compositions. Due to the high fidelity and versatility, complete conversion of the fatty acid substrates into multiple phospholipid species is achieved simultaneously, leading to membrane expansion as a first step towards a synthetic minimal cell.

    更新日期:2017-09-19
  • Managing the SOS Response for Enhanced CRISPR-Cas-based Recombineering in E. coli Through Transient Inhibition of Host RecA Activity
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-15
    Eirik Adim Moreb, Benjamin Hoover, Adam Yaseen, Nisakorn Valyasevi, Zoe Roecker, Romel Menacho-Melgar, Michael Lynch

    Phage-derived “recombineering” methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well mutation rates through the induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are co-expressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10,000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologs may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

    更新日期:2017-09-15
  • Cell-free protein synthesis enhancement from real-time NMR metabolite kinetics: redirecting energy fluxes in hybrid RRL systems
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-15
    Baptiste Panthu, Théophile Ohlmann, Johan Perrier, Uwe Schlattner, Pierre Jalinot, Bénédicte Elena-Herrmann, Gilles J. P. Rautureau

    A counter-intuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity is ceiled at native ribosome concentration and that lower amounts of ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

    更新日期:2017-09-15
  • Leaf LIMS: A Flexible Laboratory Information Management System with a Synthetic Biology Focus
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-13
    Thomas Craig, Richard Holland, Rosalinda D’Amore, James R. Johnson, Hannah V. McCue, Anthony West, Valentin Zulkower, Hille Tekotte, Yizhi Cai, Daniel Swan, Robert P Davey, Christiane Hertz-Fowler, Anthony Hall, Mark Caddick
    更新日期:2017-09-13
  • Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-12
    Zhenlin Fan, Jian Song, Tuchen Guan, Xiuxiu Lv, Jingyan Wei
    更新日期:2017-09-12
  • Cloning and transplantation of the Mesoplasma florum genome
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-11
    Vincent Baby, Fabien Labroussaa, Joëlle Brodeur, Dominick Matteau, Géraldine Gourgues, Carole Lartigue, Sebastien Rodrigue

    Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by increased phylogenetic distance between the genome donor and the recipient cell. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.

    更新日期:2017-09-12
  • Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-12
    Meng-Ying Wu, Li-Yu Sung, Hung Li, Chun-Hung Huang, Yu-Chen Hu
    更新日期:2017-09-12
  • Strategies for editing virulent staphylococcal phages using CRISPR-Cas10
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-08
    S.M. Nayeemul Bari, Forrest C. Walker, Katie Cater, Barbaros Aslan, Asma Hatoum-Aslan

    Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multi-subunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. Based on this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

    更新日期:2017-09-08
  • The Experiment Data Depot: A Web-Based Software Tool for Biological Experimental Data Storage, Sharing, and Visualization
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-08
    William C. Morrell, Garrett W. Birkel, Mark Forrer, Teresa Lopez, Tyler W. H. Backman, Michael Dussault, Christopher J. Petzold, Edward E. K. Baidoo, Zak Costello, David Ando, Jorge Alonso-Gutierrez, Kevin W. George, Aindrila Mukhopadhyay, Ian Vaino, Jay D. Keasling, Paul D. Adams, Nathan J. Hillson, Hector Garcia Martin
    更新日期:2017-09-08
  • Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive DNA Labeling and Endogenous Gene Regulation
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-07
    Shipeng Shao, Lei Chang, Yuao Sun, Yingping Hou, Xiaoying Fan, Yujie Sun
    更新日期:2017-09-07
  • Maltose Utilization as a Novel Selection Strategy for Continuous Evolution of Microbes with Enhanced Metabolite Production
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-06
    Shu-De Liu, Yi-Nan Wu, Tian-Min Wang, Chong Zhang, Xin-Hui Xing
    更新日期:2017-09-07
  • Leaf LIMS: A flexible laboratory information management system with a synthetic biology focus
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-06
    Thomas Craig, Richard Holland, Rosalinda D’Amore, James Johnson, Hannah V McCue, Anthony West, Valentin Zulkower, Hille Tekotte, Yizhi Cai, Daniel Swan, Robert P Davey, Christiane Hertz-Fowler, Anthony Hall, Mark Caddick

    This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project’s inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most LIMS systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence it enables highly configurable task based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT licence. It is freely available for download at https://leaflims.github.io.

    更新日期:2017-09-07
  • An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-06
    Lauren R. Polstein, Mark Juhas, Gabi Hanna, Nenad Bursac, Charles A. Gersbach
    更新日期:2017-09-06
  • The Timing of Transcriptional Regulation in Synthetic Gene Circuits
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-05
    Yu-Yu Cheng, Andrew J. Hirning, Krešimir Josić, Matthew R. Bennett
    更新日期:2017-09-06
  • Rapid Isolation of Centromeres from Scheffersomyces stipitis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-05
    Mingfeng Cao, Arun Somwarpet Seetharam, Andrew Josef Severin, Zengyi Shao
    更新日期:2017-09-06
  • Genetic design via combinatorial constraint specification
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-05
    Swapnil Bhatia, Michael Smanski, Christopher A. Voigt, Douglas Densmore

    We present a formal language for specifying via constraints a “design space” of DNA constructs composed of genetic parts, and an algorithm for automatically and correctly creating a novel representation of the space of satisfying designs. The language is simple, captures a large class of design spaces, and possesses algorithms for common operations on design spaces. The flexibility of this approach is demonstrated using a 16-gene nitrogen fixation pathway and genetic logic circuits.

    更新日期:2017-09-06
  • Single-Cell Microfluidics to Study the Effects of Genome Deletion on Bacterial Growth Behavior
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-05
    Xiaofei Yuan, Jillian M. Couto, Andrew Glidle, Yanqing Song, William Sloan, Huabing Yin
    更新日期:2017-09-05
  • A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-01
    Jeffrey M. Kubiak, Matthew J. Culyba, Monica Yun Liu, Charlie Y. Mo, Mark Goulian, Rahul M. Kohli
    更新日期:2017-09-04
  • Construction of Recombinant Pdu Metabolosome Shells for Small Molecule Production in Corynebacterium glutamicum
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-01
    Isabel Huber, David J. Palmer, Kira N. Ludwig, Ian R. Brown, Martin J. Warren, Julia Frunzke
    更新日期:2017-09-04
  • Engineering Isoprene Synthase Expression and Activity in Cyanobacteria
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-31
    Julie E. Chaves, Paloma Rueda-Romero, Henning Kirst, Anastasios Melis
    更新日期:2017-09-04
  • Efficient expression of glutathione peroxidase with chimeric tRNA in amber-less Escherichia coli
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-03
    Zhenlin Fan, Jian Song, Tuchen Guan, Xiuxiu Lv, JingYan Wei

    The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed based on tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%.Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

    更新日期:2017-09-04
  • Versatility of prolyl oligopeptidase B in peptide macrocyclization
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-02
    Robert Michael Sgambelluri, Miranda O Smith, Jonathan D Walton

    Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35-mer precursor peptides which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7 to 16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including D-Ala, β-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when L-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.

    更新日期:2017-09-04
  • Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-30
    Meng-Ying Wu, Li-Yu Sung, Hung Li, Chun-Hung Huang, Yu-Chen Hu

    Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g. 1,4-BDO).

    更新日期:2017-08-31
  • Corynebacterium glutamicum Chassis C1*: Building and Testing a Novel Platform Host for Synthetic Biology and Industrial Biotechnology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-30
    Meike Baumgart, Simon Unthan, Ramona Kloß, Andreas Radek, Tino Polen, Niklas Tenhaef, Moritz Fabian Müller, Andreas Küberl, Daniel Siebert, Natalie Brühl, Kay Marin, Stephan Hans, Reinhard Krämer, Michael Bott, Jörn Kalinowski, Wolfgang Wiechert, Gerd Seibold, Julia Frunzke, Christian Rückert, Volker F. Wendisch, Stephan Noack
    更新日期:2017-08-30
  • Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-29
    Shipeng Shao, Lei Chang, Yuao Sun, Yingping Hou, Xiaoying Fan, Yujie Sun

    The CRISPR/Cas9 system has made significant contribution to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current co-transfection based sgRNA co-expression systems remain inefficient and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single non-repetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its full potency, our method will facilitate the research in understanding genome structure, function and dynamics.

    更新日期:2017-08-29
  • Orthogonal Ribosome Biofirewall
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-28
    Bin Jia, Hao Qi, Bing-Zhi Li, Shuo Pan, Duo Liu, Hong Liu, Yizhi Cai, Ying-Jin Yuan
    更新日期:2017-08-28
  • Single-cell microfluidics to study the effects of genome deletion on bacterial growth behaviour
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-26
    Xiaofei Yuan, Jillian M. Couto, Andrew Glidle, Yanqing Song, William Sloan, Huabing Yin

    By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and non-growing cells) of the clean genome population was also lower. After random mutations induced by a glucose starvation treatment in chemostat culture, the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased. However, the wild type mutants retained a high diversity of growth phenotype. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

    更新日期:2017-08-28
  • The timing of transcriptional regulation in synthetic gene circuits
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-25
    Yu-Yu Cheng, Andrew John Hirning, Kresimir Josic, Matthew R Bennett

    Transcription factors and their target promoters are central to synthetic biology. By arranging these components into novel gene regulatory circuits, synthetic biologists have been able to create a wide variety of phenotypes, including bistable switches, oscillators, and logic gates. However, transcription factors (TFs) do not instantaneously regulate downstream targets. After the gene encoding a TF is turned on, the gene must first be transcribed, the transcripts must be translated, and sufficient TF must accumulate in order to bind operator sites of the target promoter. The time to complete this process, here called the “signaling time,” is a critical aspect in the design of dynamic regulatory networks, yet it remains poorly characterized. In this work, we measured the signaling time of two TFs in Escherichia coli commonly used in synthetic biology: the activator AraC and the repressor LacI. We found that signaling times can range from a few to tens of minutes, and are affected by the expression rate of the TF. Our single-cell data also show that the variability of the signaling time increases with its mean. To validate these signaling time measurements, we constructed a two-step genetic cascade, and showed that the signaling time of the full cascade can be predicted from those of its constituent steps. These results provide concrete estimates for the timescales of transcriptional regulation in living cells, which are important for understanding the dynamics of synthetic transcriptional gene circuits.

    更新日期:2017-08-26
  • Maltose Utilization as a Novel Selection Strategy for Continuous Evolution of Microbes with Enhanced Metabolite Production
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-25
    Shu-De Liu, Yi-Nan Wu, Tian-Min Wang, Chong Zhang, Xin-Hui Xing

    We have developed a novel selection circuit based on carbon source utilization that establishes and sustains growth-production coupling over several generations in a medium with maltose as the sole carbon source. In contrast to traditional antibiotic resistance–based circuits, we first approved that coupling of cell fitness to metabolite production by our circuit was more robust with a much lower escape risk even after many rounds of selection. We then applied the selection circuit to the optimization of L-tryptophan (L-trp) production. We demonstrated that it enriched for specific mutants with increased L-trp productivity whether applied to a small defined, or a relatively large and undefined, mutational library. From the latter, we identified four novel mutations with enhanced L-trp output. Finally, we used it to select for several high L-trp producers with randomly generated genome-wide mutations and obtained strains with up to 65% increased L-trp production. This selection circuit provides new perspectives for the optimization of microbial cell factories for diverse metabolite production and the discovery of novel genotype-phenotype associations at the single-gene and whole genome levels.

    更新日期:2017-08-26
  • Rapid Isolation of Centromeres from Scheffersomyces stipitis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-24
    Mingfeng Cao, Arun Somwarpet Seetharam, Andrew Josef Severin, Zengyi Shao

    Centromeres (CENs) are the chromosomal regions promoting kinetochore formation for faithful chromosome segregation. In yeasts, CENs have been recognized as the essential elements for extra-chromosomal DNA stabilization. However, the epigeneticity of CENs makes their localization on individual chromosomes very challenging, especially in many not well-studied nonconventional yeast species. Previously, we applied a stepwise method to identify a 500-bp CEN5 from Scheffersomyces stipitis chromosome 5 and experimentally confirmed its critical role on improving plasmid stability. Here we report a library-based strategy that integrates in silico GC3 chromosome scanning and high-throughput functional screening, which enabled the isolation of all eight S. stipitis centromeres with a 16, 000-fold reduction in sequence very efficiently. Further identification of a 125-bp CEN core sequence that appears multiple times on each chromosome but all in the unique signature GC3-valley indicates that CEN location might be accurately discerned by their local GC3 percentages in a subgroup of yeasts.

    更新日期:2017-08-25
  • Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-24
    Nicholas C. O. Lee, Jung-Hyun Kim, Nikolai S. Petrov, Hee-Sheung Lee, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, Natalay Kouprina
    更新日期:2017-08-25
  • Multiplexing Engineered Receptors for Multiparametric Evaluation of Environmental Ligands
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-23
    Rachel M. Hartfield, Kelly A. Schwarz, Joseph J. Muldoon, Neda Bagheri, Joshua N. Leonard
    更新日期:2017-08-23
  • Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    Andriy Didovyk, Taishi Tonooka, Lev Tsimring, Jeff Hasty
    更新日期:2017-08-22
  • A small-molecule inducible synthetic circuit for control of the SOS gene network without DNA damage
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    Jeffrey M Kubiak, Matthew J. Culyba, Monica Yun Liu, Charlie Y. Mo, Mark Goulian, Rahul M. Kohli

    The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes auto-proteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.

    更新日期:2017-08-22
  • Construction of recombinant Pdu metabolosome shells for small molecule production in Corynebacterium glutamicum
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    Isabel Huber, David J. Palmer, Kira N. Ludwig, Ian Brown, Martin J. Warren, Julia Frunzke

    Bacterial microcompartments have significant potential in the area of industrial biotechnology for the production of small molecules, especially involving metabolic pathways with toxic or volatile intermediates. Corynebacterium glutamicum is an established industrial workhorse for the production of amino acids and has been investigated for the production of a variety of further value-added products. Herein, we describe components for the establishment of bacterial microcompartments as production chambers in C. glutamicum. Within this study, we optimized genetic clusters for the expression of the shell components of the Citrobacter freundii propanediol utilization (Pdu) bacterial compartment, thereby facilitating heterologous compartment production in C. glutamicum. Upon induction, transmission electron microscopy images of thin sections from these strains revealed microcompartment-like structures within the cytosol. Furthermore, we demonstrate that it is possible to target eYFP to the empty microcompartments through C-terminal fusions with synthetic scaffold interaction partners (PDZ, SH3 and GBD) as well as with a non-native C-terminal targeting peptide from AdhDH (Klebsiella pneumonia). Thus, we show that it is possible to target proteins to compartments where N-terminal targeting is not possible. The overproduction of PduA alone leads to the construction of filamentous structures within the cytosol and eYFP molecules are localized to these structures when they are N-terminally fused to the P18 and D18 encapsulation peptides from PduP and PduD, respectively. In future, these nanotube-like structures might be used as scaffolds for directed cellular organization and pathway enhancement.

    更新日期:2017-08-22
  • The Experiment Data Depot: a web-based software tool for biological experimental data storage, sharing, and visualization
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    William Morrell, Garrett Birkel, Mark Forrer, Teresa Lopez, Tyler Backman, Michael Dussault, Christopher J. Petzold, Edward E.K. Baidoo, Zak Costello, David Ando, Jorge Alonso Gutierrez, Kevin George, Aindrila Mukhopadhyay, Ian Vaino, Jay D Keasling, Paul D. Adams, Nathan J Hillson, Hector Garcia Martin

    Although recent advances in synthetic biology allow us to produce biological designs more efficiently than ever, our ability to predict the end result of these designs is still nascent. Predictive models require large amounts of high-quality data to be parametrized and tested, which are not generally available. Here, we present the Experiment Data Depot (EDD), an online tool designed as a repository of standardized experimental data and metadata. EDD provides a convenient way to upload a variety of data types, visualize these data, and export them in a standardized fashion for use with predictive algorithms. In this paper, we describe EDD and showcase its utility for three different use cases: storage of characterized synthetic biology parts, leveraging proteomics data to improve biofuel yield, and the use of extracellular metabolite concentrations to predict intracellular metabolic fluxes.

    更新日期:2017-08-22
  • Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    José M. Duarte, Içvara Barbier, Yolanda Schaerli
    更新日期:2017-08-21
  • NOT Gate Genetic Circuits to Control Gene Expression in Cyanobacteria
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-21
    Arnaud Taton, Amy T. Ma, Mizuho Ota, Susan S. Golden, James W. Golden
    更新日期:2017-08-21
  • Controlling Bdellovibrio bacteriovorus Gene Expression and Predation Using Synthetic Riboswitches
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-18
    Mohammed Dwidar, Yohei Yokobayashi
    更新日期:2017-08-19
  • Production of Functional Anti-Ebola Antibodies in Pichia pastoris
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-17
    Oliver Purcell, Patrick Opdensteinen, William Chen, Ky Lowenhaupt, Alexander Brown, Mario Hermann, Jicong Cao, Niklas Tenhaef, Eric Kallweit, Robin Kastilan, Anthony J. Sinskey, Pablo Perez-Pinera, Johannes F. Buyel, Timothy K. Lu
    更新日期:2017-08-17
  • Yeast Terminator Function Can Be Modulated and Designed on the Basis of Predictions of Nucleosome Occupancy
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-17
    Nicholas J. Morse, Madan R. Gopal, James M. Wagner, Hal S. Alper
    更新日期:2017-08-17
  • Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-17
    Fei Chen, Seraphine V. Wegner
    更新日期:2017-08-17
  • Controlling Bdellovibrio bacteriovorus Gene Expression and Predation Using Synthetic Riboswitches
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-16
    Mohammed Dwidar, Yohei Yokobayashi

    Bdellovibrio bacteriovorus is a predatory bacterium that feeds on Gram-negative bacteria including a wide range of pathogens and thus has potential applications as a biocontrol agent. Owing to its unique life cycle, however, there are limited tools that enable genetic manipulation of B. bacteriovorus. This work describes our first steps towards engineering the predatory bacterium for practical applications by developing basic genetic parts to control gene expression. Specifically, we evaluated four robust promoters that are active during the attack phase of B. bacteriovorus. Subsequently, we tested several synthetic riboswitches that have been reported to function in Escherichia coli, and identified theophylline-activated riboswitches that function in B. bacteriovorus. Finally, we inserted the riboswitch into the bacterial chromosome to regulate expression of the flagellar sigma factor fliA which was previously predicted to be essential for predation, and observed that the engineered strain shows a faster predation kinetics in the presence of theophylline.

    更新日期:2017-08-16
  • A Tug-of-War Mechanism for Pattern Formation in a Genetic Network
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-15
    Marcella M. Gomez, Murat Arcak
    更新日期:2017-08-15
  • Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-15
    Isha Singh, Myong-Jung Kim, Robert W. Molt, Shuichi Hoshika, Steven A. Benner, Millie M. Georgiadis
    更新日期:2017-08-15
  • Adoption of the Q Transcriptional System for Regulating Gene Expression in Stem Cells
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    Michael Fitzgerald, Chelsea Gibbs, Adrian A. Shimpi, Tara L. Deans
    更新日期:2017-08-15
  • Bacterial microcolonies in gel beads for high-throughput screening of libraries in synthetic biology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    José M. Duarte, Içvara Barbier, Yolanda Schaerli

    Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g. being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g. expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

    更新日期:2017-08-14
  • Blue light switchable bacterial adhesion as a key step towards the design of biofilms
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    Fei Chen, Seraphine Valeska Wegner

    The control of where and when bacterial adhere to a substrate is a key step towards controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E.coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tuneable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.

    更新日期:2017-08-14
  • NOT gate genetic circuits to control gene expression in cyanobacteria
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    Arnaud Taton, Amy T Ma, Mizuho Ota, Susan Golden , James W. Golden

    To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches. Four NOT gates were tested and characterized in five cyanobacterial strains using fluorescent reporter-gene assays. In comparison to alternative systems used to downregulate gene expression in cyanobacteria, these NOT gates performed well, reducing YFP reporter expression by 4 to 50-fold. We further evaluated these NOT gates by controlling the expression of the ftsZ gene, which encodes a prokaryotic tubulin homolog that is required for cell division and is essential for Synechococcus elongatus PCC 7942. These NOT gates would facilitate cyanobacterial genetic engineering or the study of essential cellular processes.

    更新日期:2017-08-14
  • Corynebacterium glutamicum chassis C1*: Building and testing a novel platform host for synthetic biology and industrial biotechnology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    Meike Baumgart, Simon Unthan, Ramona Kloss, Andreas Radek, Tino Polen, Niklas Tenhaef, Moritz Fabian Müller, Andreas Küberl, Daniel Siebert, Natalie Brühl, Kay Marin, Stephan Hans, Reinhard Krämer, Michael Bott, Joern Kalinowski, Wolfgang Wiechert, Gerd Seibold, Julia Frunzke, Christian Rückert, Volker Wendisch, Stephan Noack

    Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with D-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.

    更新日期:2017-08-14
  • Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-14
    Carlos A. Cruz-Teran, Karthik Tiruthani, Adam Mischler, Balaji M. Rao
    更新日期:2017-08-14
  • Method to assemble genomic DNA fragments or genes on Human Artificial Chromosome with regulated kinetochore using a multi-integrase system
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-11
    Nicholas C.O. Lee, Jung-Hyun Kim, Nikolai Petrov, Hee-sheung Lee, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, Natalay Kouprina

    The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: i) Cloning of large genomic segments; ii) Insertion of multiple DNA blocks at a precise location and iii) The capability to eliminate the assembled region from cells. In this study, we designed the Iterative Integration System (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with Human Artificial Chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes: The potential to assemble an unlimited number of genomic DNA segments; A small (<60bp) scar sites between adjacent DNA segments, allowing genes reassembled from segments to be spliced correctly; A marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; Presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoidtetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its’ pre-transformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoidtetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

    更新日期:2017-08-11
  • Rational Modular RNA Engineering Based on In Vivo Profiling of Structural Accessibility
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-10
    Abigail N. Leistra, Paul Amador, Aishwarya Buvanendiran, Alex Moon-Walker, Lydia M. Contreras
    更新日期:2017-08-11
  • Rapid and scalable preparation of bacterial lysates for cell-free gene expression
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-10
    Andriy Didovyk, Taishi Tonooka, Lev Tsimring, Jeff Hasty

    Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive beta-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

    更新日期:2017-08-10
  • A Microfluidic Biodisplay
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-10
    Francesca Volpetti, Ekaterina Petrova, Sebastian J. Maerkl
    更新日期:2017-08-10
  • Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-10
    Sungho Jang, Sungyeon Jang, Yu Xiu, Taek Jin Kang, Sang-Hyeup Lee, Mattheos A. G. Koffas, Gyoo Yeol Jung
    更新日期:2017-08-10
  • Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-10
    Drew M. DeLorenzo, William R. Henson, Tae Seok Moon
    更新日期:2017-08-10
  • An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-08-09
    Lauren Polstein, Mark Juhas, Gabi Hanna, Nenad Bursac, Charles A. Gersbach

    The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic “on” switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.

    更新日期:2017-08-10
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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