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ACS Synthetic Biology

Joao Cardoso, Kristian Jensen, Christian Lieven, Anne Sofie Lærke Hansen, Svetlana Galkina, Moritz Emanuel Beber, Emre Özdemir, Markus Herrgard, Henning Redestig, Nikolaus Sonnenschein
Computational systems biology methods enable rational design of cell factories on a genome-scale and thus accelerate the engineering of cells for the production of valuable chemicals and proteins. Unfortunately, for the majority of these methods' implementations are either not published, rely on proprietary software, or do not provide documented interfaces, which has precluded their mainstream adoption in the field. In this work we present cameo, a platform-independent software that enables in silico design of cell factories and targets both experienced modelers as well as users new to the field...
March 20, 2018: ACS Synthetic Biology
Ciarán L Kelly, George M Taylor, Andrew Hitchcock, Antonio Torres-Méndez, John T Heap
Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light...
March 15, 2018: ACS Synthetic Biology
Suhyung Cho, Donghui Choe, Eunju Lee, Sun Chang Kim, Bernhard Ø Palsson, Byung-Kwan Cho
Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq)...
March 15, 2018: ACS Synthetic Biology
Eric Poliner, Tomomi Takeuchi, Zhi-Yan Du, Christoph Benning, Eva Farre
Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalga lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA...
March 8, 2018: ACS Synthetic Biology
Mathieu C Husser, Philippe Q N Vo, Hugo Sinha, Fatemeh Ahmadi, Steve C C Shih
The expression of a recombinant gene in a host organism through induction can be an extensively manual and labor-intensive procedure. Several methods have been developed to simplify the protocol, but none has fully replaced the traditional IPTG-based induction. To simplify this process, we describe the development of an autoinduction platform based on digital microfluidics. This system consists of a 600 nm LED and a light sensor to enable the real-time monitoring of  the optical density (OD) samples coordinated with the semicontinuous mixing of a bacterial culture...
March 8, 2018: ACS Synthetic Biology
V Siddartha Yerramilli, Kyung Hyuk Kim
RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells...
March 7, 2018: ACS Synthetic Biology
Takumi Furusatoa, Fumihiro Horie, Hideaki T Matsubayashi, Kazuaki Amikura, Yutetsu Kuruma, Takuya Ueda
Cell division is the most dynamic event in the cell cycle. Recently, efforts have been made to reconstruct it using the individual component proteins to obtain a better understanding of the process of self-reproduction of cells. However, such reconstruction studies are frequently hampered by difficulties in preparing membrane-associated proteins. Here we demonstrate a de novo synthesis approach based on a cell-free translation system. Genes for fundamental cell division proteins, FtsZ, FtsA, and ZipA, were expressed inside the lipid compartment of giant vesicles (GVs)...
March 7, 2018: ACS Synthetic Biology
Marc Biarnes, Chang-Kwon Lee, Takuya Nihira, Rainer Breitling, Eriko Takano
Chemically inducible transcription factors are widely used to control gene expression of synthetic devices. The bacterial quorum sensing system is a popular tool to achieve such control. However, different quorum sensing systems have been found to cross-talk, both between themselves and with the hosts of these devices, and they are leaky by nature. Here we evaluate the potential use of the γ-butyolactone system from Streptomyces coelicolor A3(2) M145 as a complementary regulatory circuit. First, two additional genes responsible for the biosynthesis of γ-butyrolactones were identified in S...
March 6, 2018: ACS Synthetic Biology
Nicolas Krink-Koutsoubelis, Anne Christina Loechner, Anna Lechner, Hannes Link, Charles M Denby, Bastian Voegeli, Tobias J Erb, Satoshi Yuzawa, Tadas Jakociunas, Leonard Katz, Michael K Jensen, Victor Sourjik, Jay D Keasling
Short chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S...
March 2, 2018: ACS Synthetic Biology
Jonathan Naylor, Harold Fellermann, Yuchun Ding, Waleed K Mohammed, Nicholas S Jakubovics, Felix Dafhnis-Calas, Stephan Heeb, Miguel Cámara, Joy Mukherjee, Catherine A Biggs, Phillip C Wright, Natalio Krasnogor
No abstract text is available yet for this article.
March 1, 2018: ACS Synthetic Biology
Nhung T Nguyen, Lian He, Margie Martinez-Moczygemba, Yun Huang, Yubin Zhou
Tools capable of modulating gene expression in living organisms is very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically-inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically-encoded calcium actuators with dCas9 to control gene expression...
February 28, 2018: ACS Synthetic Biology
Ellis Whitehead, Fabian Rudolf, Hans-Michael Kaltenbach, Jörg Stelling
Robotic automation in synthetic biology is especially relevant for liquid handling to facilitate complex experiments. However, research tasks that are not highly standardized are still rarely automated in practice. Two main reasons for this are the substantial investments required to translate molecular biological protocols into robot programs, and the fact that the resulting programs are often too specific to be easily re-used and shared. Recent developments of standardized protocols and dedicated programming languages for liquid-handling operations addressed some aspects of ease-of-use and portability of protocols...
February 27, 2018: ACS Synthetic Biology
Xin-Yuan Qiu, Lv-Yun Zhu, Chu-Shu Zhu, Jia-Xin Ma, Tao Hou, Xiao-Min Wu, Si-Si Xie, Lu Min, De-An Tan, Dongyi Zhang, Lingyun Zhu
MicroRNAs have been reported related to multiple diseases and has potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost and low sensitivity of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on Rolling Circle Amplification, CRISPR-Cas9, and split-Horseradish Peroxidase techniques...
February 27, 2018: ACS Synthetic Biology
Hidetoshi Teramoto, Yoshimi Amano, Fumie Iraha, Katsura Kojima, Takuhiro Ito, Kensaku Sakamoto
The genetic code in bacteria and animal cells has been expanded to incorporate novel amino acids into proteins. Recent efforts have enabled genetic code expansion in nematodes, flies, and mice, whereas such engineering is rare with industrially useful animals. In the present study, we engineered the silkworm Bombyx mori to synthesize silk fiber functionalized with azidophenylalanine. For this purpose, we developed a bacterial system to screen for B. mori phenylalanyl-tRNA synthetases with altered amino-acid specificity...
February 26, 2018: ACS Synthetic Biology
Joseph A Harvey, Laura S Itzhaki, Ewan Main
Harnessing and controlling self-assembly is an important step in developing proteins as novel biomaterials. With this goal, here we report the design of a general genetically programmed system that covalently concatenates multiple distinct protein domains into specific assembled arrays. It is driven by iterative intein-mediated Native Chemical Ligation (NCL) under mild native conditions. The system uses a series of initially inert recombinant protein fusions that sandwich the protein modules to be ligated between one of a number of different affinity tags and an intein protein domain...
February 23, 2018: ACS Synthetic Biology
Sha Hou, Qin Qin, Junbiao Dai
Yeast can be used as a microbial cell factory to produce valuable chemicals. However, introducing an exogenous pathway into particular or different chromosomal locations for stable expression is still a daunting task. To address this issue, we designed a DNA cassette called a "wicket", which can be integrated into the yeast genome at designated loci to accept exogenous DNA upon excision by a nuclease. Using this system, we demonstrated that, in strains with "wickets", we could achieve near 100% efficiency for integration of the β-carotene pathway with no need for selective markers...
February 23, 2018: ACS Synthetic Biology
Anne Pihl Bali, Hans Jasper Genee, Morten Sommer
Understanding and engineering solute transporters is important for metabolic engineering and the development of therapeutics. However, limited available experimental data on membrane transporters makes sequence-function relationships complex to predict. Here we apply a ligand-responsive biosensor systems that enable selective growth of E. coli cells only if they functionally express an importer that is specific to the biosensor ligand. Using this system in a directed evolution framework, we successfully engineer the specificity of nicotinamide riboside transporters, PnuC, to accept thiamine as a substrate...
February 23, 2018: ACS Synthetic Biology
Ana I Benítez-Mateos, Irantzu Llarena, Ana Sánchez-Iglesias, Fernando López Gallego
Fabrication of protein-based biomaterials is an arduous and time-consuming procedure with multiple steps. In this work, we describe a portable tool-kit that integrates both cell-free protein synthesis (CFPS) and protein immobilization in one-pot, just by mixing DNA, solid materials and a CFPS system. We construct a modular set of plasmids that fuse the N-terminus of superfolded Green Fluorescent Protein (sGFP) with different peptide-tags (poly-Cys, poly-His and poly-Lys), which drive the immobilization of the protein on the tailored material (agarose beads with different functionalities, gold nanorods and silica nanoparticles)...
February 23, 2018: ACS Synthetic Biology
Soumya Kannan, Thomas Sams, Jérôme Maury, Christopher T Workman
Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process...
February 19, 2018: ACS Synthetic Biology
Cheng Zhao, YongKyoung Kim, Yining Zeng, Man Li, Xin Wang, Cheng Hu, Connor Gorman, Susie Y Dai, Shi-You Ding, Joshua S Yuan
Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene...
February 16, 2018: ACS Synthetic Biology
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