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ACS Synthetic Biology

Andrew Lerner, Hayretin Yumerefendi, Odessa Goudy, Brian D Strahl, Brian Kuhlman
Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus...
November 15, 2018: ACS Synthetic Biology
Gourab Chatterjee, Yuan-Jyue Chen, Georg Seelig
Strand displacement reactions are widely used in DNA nanotechnology as a building block for engi-neering molecular computers and machines. Here, we demonstrate that strand displacement-based probes can be triggered by RNA expressed in mammalian cells, thus taking a step towards adapt-ing the DNA nanotechnology toolbox to a cellular environment. We systematically compare different probe architectures in order to identify a design that works robustly in living cells. Our optimized strand displacement probe combines chemically modified nucleic acids that enhance stability to degradation by cellular nucleases with structural elements that improve probe retention in the cytoplasm...
November 15, 2018: ACS Synthetic Biology
William Black, Edward King, Yixi Wang, Ana Jenic, Andrew T Rowley, Kosuke Seki, Ray Luo, Han Li
The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ~10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus...
November 15, 2018: ACS Synthetic Biology
Kai-Hong Guo, Kun-Hua Lu, Yi-Chun Yeh
Despite the importance of 2-phenylacetic acid, a plant hormone in the endogenous auxin family, its biosynthesis pathway has yet to be elucidated. In this study, we developed a novel whole-cell biosensor for the simultaneous quantification of 2-phenylacetic acid (PA) and 2-phenylethylamine (PEA) through the regulation of bacterial catabolism of aromatic compounds. We used the PA regulon to enable the recognition of PA and PEA. Differentiation of PEA from PA involves the incorporation of the FeaR regulon within the same whole-cell biosensor to report the presence of aromatic amines...
November 15, 2018: ACS Synthetic Biology
Jae Seong Lee, Jin Hyoung Park, Tae Kwang Ha, Mojtaba Samoudi, Nathan E Lewis, Bernhard O Palsson, Helene Faustrup Kildegaard, Gyun Min Lee
Generation of recombinant Chinese hamster ovary (rCHO) cell lines is critical for the production of therapeutic proteins. However, the high degree of phenotypic heterogeneity among generated clones, referred to as clonal variation, makes the rCHO cell line development process inefficient and unpredictable. Here, we investigated the major genomic causes of clonal variation. We found the following: (1) consistent with previous studies, a strong variation in rCHO clones in response to hypothermia (33 vs 37 °C) after random transgene integration; (2) altered DNA sequence of randomly integrated cassettes, which occurred during the integration process, affecting the transgene expression level in response to hypothermia; (3) contrary to random integration, targeted integration of the same expression cassette, without any DNA alteration, into three identified integration sites showed the similar response of transgene expression in response to hypothermia, irrespective of integration site; (4) switching the promoter from CMV to EF1α eliminated the hypothermia response; and (5) deleting the enhancer part of the CMV promoter altered the hypothermia response...
November 14, 2018: ACS Synthetic Biology
Bryan Bartley, Kiri Choi, Meher Samineni, Zach Zundel, Tramy Nguyen, Chris J Myers, Herbert Sauro
This paper presents pySBOL, a software library for computer-aided design of synthetic biological systems in the Python scripting language. This library provides an easy-to-use, object-oriented, application programming interface (API) with low barrier of entry for synthetic biology application developers. The pySBOL library enables reuse of genetic parts and designs through standardized data exchange with biological parts repositories and software tools that communicate using the Synthetic Biology Open Language (SBOL)...
November 13, 2018: ACS Synthetic Biology
Dominik Beuter, José Vicente Gomes-Filho, Lennart Randau, Francisco Díaz-Pascual, Knut Drescher, Hannes Link
Construction of pooled genetic variant libraries has become very fast and versatile. The current limitation of this technique is to select cells with a desired phenotype from very large libraries. Especially cells with poor fitness and slow growth are difficult to select because they are rapidly outcompeted by fitter cells. Here, we demonstrate selective and high-throughput enrichment of slow-growing strains using a fluorescent TIMER protein and flow cytometry. As a proof of principle, we created a metabolism-wide CRISPR interference library for Escherichia coli and enriched targets that interfere with amino acid metabolism...
November 13, 2018: ACS Synthetic Biology
Noa Katz, Roni Cohen, Oz Solomon, Beate Kaufmann, Orna Atar, Zohar Yakhini, Sarah Goldberg, Roee Amit
We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region...
November 13, 2018: ACS Synthetic Biology
Hiroya Itoh, Makoto Matsui, Yuki Miyamura, Itaru Takeda, Jun Ishii, Toshitaka Kumagai, Masayuki Machida, Takashi Shibata, Masanori Arita
For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes...
November 13, 2018: ACS Synthetic Biology
Naveen Venayak, Kaushik Raj, Rohil Jaydeep, Radhakrishnan Mahadevan
Metabolic engineers aim to genetically modify microorganisms to improve their ability to produce valuable compounds. Despite the prevalence of growth-coupled production processes, these strategies can significantly limit production rates. Instead, rates can be improved by decoupling and optimizing growth and production independently, and operating with a growth stage followed by a production stage. Here, we implement a bistable transcriptional controller to decouple and switch between these two states. We optimize the controller in anaerobic conditions, typical of industrial fermentations, to ensure stability and tight expression control, while improving switching dynamics...
November 12, 2018: ACS Synthetic Biology
Christopher M Dundas, Austin J Graham, Dwight K Romanovicz, Benjamin K Keitz
The relative scarcity of well-defined genetic and metabolic linkages to material properties impedes biological production of inorganic materials. The physiology of electroactive bacteria is intimately tied to inorganic transformations, which makes genetically tractable and well-studied electrogens, such as Shewanella oneidensis, attractive hosts for material synthesis. Notably, this species is capable of reducing a variety of transition-metal ions into functional nanoparticles, but exact mechanisms of nanoparticle biosynthesis remain ill-defined...
November 9, 2018: ACS Synthetic Biology
Ziwei Zhong, Arjun Ravikumar, Chang C Liu
We recently developed an orthogonal DNA replication (OrthoRep) system capable of driving the rapid continuous evolution of genes in vivo. However, OrthoRep uses a special transcription system whose components ( e.g. promoters) have previously limited the strength with which OrthoRep-encoded genes can be expressed. Here, we report a collection of synthetic or evolved OrthoRep expression parts that allow OrthoRep-encoded genes to span expression levels matching those of endogenous Saccharomyces cerevisiae genes...
November 8, 2018: ACS Synthetic Biology
Mary Ann Madsen, Stefan Semerdzhiev, Anna Amtmann, Thierry Tonon
The genetic engineering of microbial cell factories is a sustainable alternative to the chemical synthesis of organic compounds. Successful metabolic engineering often depends on manipulating several enzymes, requiring multiple transformation steps and selection markers, as well as protein assembly and efficient substrate channeling. Naturally occurring fusion genes encoding two or more enzymatic functions may offer an opportunity to simplify the engineering process and to generate ready-made protein modules, but their functionality in heterologous systems remains to be tested...
November 8, 2018: ACS Synthetic Biology
Maaruthy Yelleswarapu, Ardjan van der Linden, Bob van Sluijs, Pascal Pieters, Emilien Dubuc, Tom de Greef, Wilhelm T S Huck
Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterised toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behaviour are scarce. Here, we present a genetic oscillator implemented in an E.coli based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif which utilizes the native transcriptional machinery of E...
November 8, 2018: ACS Synthetic Biology
William Waites, Goksel Misirli, Matteo Cavaliere, Vincent Danos, Anil Wipat
A central strategy of synthetic biology is to understand the basic processes of living creatures through engineering organisms using the same building blocks. Biological machines described in terms of parts can be studied by computer simulation in any of several languages or robotically assembled in vitro. In this paper we present a language, the Genetic Circuit Description Language (GCDL) and a compiler, the Genetic Circuit Compiler (GCC). This language describes genetic circuits at a level of granularity appropriate both for automated assembly in the laboratory and deriving simulation code...
November 8, 2018: ACS Synthetic Biology
Dennis Hell
The cytokines tumor necrosis factor α (TNFα) and interleukin 1 β (IL-1β) are both strong NF-κB activators and some of the first cytokines to be released in an inflammatory process. TNFα and IL-1β are present in many autoimmune diseases, such as rheumatoid arthritis (RA). TNFα and IL-1β-blocking therapies are quite successful and established in the treatment of RA, but may also be promising in other diseases. For the treatment of recurring autoimmune diseases, strong controlled sensor-effector cells inhibiting TNFα or IL-1β appear highly predestined...
November 8, 2018: ACS Synthetic Biology
James Alastair McLaughlin, Chris J Myers, Zach Zundel, Nathan Wilkinson, Christian Atallah, Anil Wipat
The Synthetic Biology Open Language (SBOL) is a data standard for the in silico representation of biological designs, such as engineered genetic circuits and their constituent DNA and protein components. The SBOL specification is implemented in the form of software libraries, which can then be used to add SBOL support to both new and existing software tools. Examples of existing SBOL libraries include libSBOLj for Java, libSBOL for C, and pySBOL for Python. These libraries can be used to develop software that runs on a server or is installed locally on a computer...
November 7, 2018: ACS Synthetic Biology
Premkumar Jayaraman, Jing Wui Yeoh, Jingyun Zhang, Chueh Loo Poh
To program cells in a dynamic manner, synthetic biologists require precise control over the threshold levels and timing of gene expression. However, in practice, modulating gene expression is widely carried out using prototypical ligand-inducible promoters, which have limited tunability and spatiotemporal resolution. Here, we built two dual-input hybrid promoters, each retaining the function of the ligand-inducible promoter while being enhanced with a blue-light-switchable tuning knob. Using the new promoters, we show that both ligand and light inputs can be synchronously modulated to achieve desired amplitude or independently regulated to generate desired frequency at a specific amplitude...
November 6, 2018: ACS Synthetic Biology
Ann-Kathrin Löbs, Cory Schwartz, Sarah Thorwall, Ian Wheeldon
The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity...
November 5, 2018: ACS Synthetic Biology
Yangyang Shao, Ning Lu, Zhongjun Qin, Xiaoli Xue
Eukaryotic cells usually contain multiple linear chromosomes. Recently, we artificially created a functional single-chromosome yeast via sequential two-chromosome fusion utilizing the high performance of the CRISPR-Cas9 system and homologous recombination in Saccharomyces cerevisiae. In this paper, we adapted this method for the simultaneous fusion of multiple chromosomes. We demonstrated the fusion of two, two-chromosome sets with a 75% positive rate and three-chromosome fusions with a 50% positive rate. We also found that by using an additional selection marker, the positive rate of two-chromosome fusions reached 100%...
November 5, 2018: ACS Synthetic Biology
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