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ACS Synthetic Biology

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...
September 15, 2017: ACS Synthetic Biology
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...
September 15, 2017: ACS Synthetic Biology
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 (tRNA(UTu)) 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...
September 12, 2017: ACS Synthetic Biology
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...
September 11, 2017: ACS Synthetic Biology
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...
September 8, 2017: ACS Synthetic Biology
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...
September 6, 2017: ACS Synthetic Biology
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 proved 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...
September 6, 2017: ACS Synthetic Biology
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.
September 5, 2017: ACS Synthetic Biology
Yu-Yu Cheng, Andrew J Hirning, Krešimir Josić, 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...
September 5, 2017: ACS Synthetic Biology
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...
September 5, 2017: ACS Synthetic Biology
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...
September 2, 2017: ACS Synthetic Biology
Isabel Huber, David J Palmer, Kira N Ludwig, Ian R 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 diamines, dicarboxylic acids, polymers and biobased fuels. Herein, we describe components for the establishment of bacterial microcompartments as production chambers in C...
September 1, 2017: ACS Synthetic Biology
Julie E Chaves, Paloma Rueda-Romero, Henning Kirst, Anastasios Melis
Efforts to heterologously produce quantities of isoprene hydrocarbons (C5H8) renewably from CO2 and H2O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins...
August 31, 2017: ACS Synthetic Biology
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...
August 30, 2017: ACS Synthetic Biology
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...
August 29, 2017: ACS Synthetic Biology
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...
August 26, 2017: ACS Synthetic Biology
Nicholas C O Lee, Jung-Hyun Kim, Nikolai S 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 a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid(tetO)-HAC)...
August 24, 2017: ACS Synthetic Biology
Rachel M Hartfield, Kelly A Schwarz, Joseph J Muldoon, Neda Bagheri, Joshua N Leonard
Engineered cell-based therapies comprise a promising, emerging biomedical technology. Broad utilization of this strategy will require new approaches for implementing sophisticated functional programs, such as sensing and responding to the environment in a defined fashion. Toward this goal, we investigated whether our self-contained receptor and signal transduction system (MESA) could be multiplexed to evaluate extracellular cues, with a focus on elucidating principles governing the integration of such engineered components...
August 23, 2017: ACS Synthetic Biology
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...
August 21, 2017: ACS Synthetic Biology
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...
August 21, 2017: ACS Synthetic Biology
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