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ACS Synthetic Biology

Bob Van Hove, Chiara Guidi, Lien De Wannemaeker, Jo Maertens, Marjan De Mey
A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process...
March 24, 2017: ACS Synthetic Biology
Mumun Gencoglu, Alexander Schmidt, Attila Becskei
Protein interaction networks play a key role in signal processing. Despite the progress in identifying the interactions, the quantification of their strengths lags behind. Here we present an approach to quantify the in vivo binding of proteins to their binding partners in signaling-transcriptional networks, by the pairwise genetic isolation of each interaction and by varying the concentration of the interacting components over time. The absolute quantification of the protein concentrations was performed with targeted mass spectrometry...
March 23, 2017: ACS Synthetic Biology
Jun Li, Wilhelm Haas, Kirsten Jackson, Erkin Kuru, Michael C Jewett, Z Hugh Fan, Steven P Gygi, George M Church
To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batch format PURE system...
March 23, 2017: ACS Synthetic Biology
Shintaro Ryo, Jun Ishii, Toshihide Matsuno, Yasuyuki Nakamura, Daiki Matsubara, Masahiro Tominaga, Akihiko Kondo
The GAL expression system is the most frequently used induction technique in the yeast Saccharomyces cerevisiae. Here we report a simple but powerful genetic circuit for use with the GAL induction system. Briefly, an artificial positive feedback circuit was incorporated into the GAL regulatory network. We selected green fluorescent protein (GFP) as a reporter of GAL1 induction, and designed a strain that expressed a constitutively active Gal3 mutant protein (Gal3(c)) under control of the GAL10 promoter. In the resulting strain, GAL1 and GAL10 promoters regulate the expression of GFP and GAL3(c), respectively...
March 21, 2017: ACS Synthetic Biology
Marie-Laurence Lemay, Denise M Tremblay, Sylvain Moineau
Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages...
March 21, 2017: ACS Synthetic Biology
Timothy A Tomko, Mary J Dunlop
Microorganisms can produce hydrocarbons that can serve as replacements or additions to conventional liquid fuels for use in the transportation sector. However, a common problem in the microbial synthesis of biofuels is that these compounds often have toxic effects on the cell. In this study, we focused on mitigating the toxicity of the bio-jet fuel precursor pinene on Escherichia coli. We used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E...
March 20, 2017: ACS Synthetic Biology
Tsz Kin Martin Tsui, Travis H Hand, Emily C Duboy, Hong Li
Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology...
March 20, 2017: ACS Synthetic Biology
Johannes Döhlemann, Marcel Wagner, Carina Happel, Martina Carrillo, Patrick Sobetzko, Tobias J Erb, Martin Thanbichler, Anke Becker
A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus...
March 20, 2017: ACS Synthetic Biology
Anum Azam Glasgow, Han Teng Wong, Danielle Tullman-Ercek
The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins...
March 16, 2017: ACS Synthetic Biology
Ulrike Obst, Timothy K Lu, Volker Sieber
Yeasts are powerful eukaryotic hosts for the production of recombinant proteins due to their rapid growth to high cell densities and ease of genetic modification. For large-scale industrial production, secretion of a protein offers the advantage of simple and efficient downstream purification that avoids costly cell rupture, denaturation and refolding. The methylotrophic yeast Pichia pastoris (Komagataella phaffi) is a well-established expression host that has the ability to perform post-translational modifications and is generally regarded as safe (GRAS)...
March 15, 2017: ACS Synthetic Biology
Viktor Stein, Masuda Nabi, Kirill Alexandrov
The rational construction of synthetic protein switches with pre-defined input-output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein-protein interactions...
March 14, 2017: ACS Synthetic Biology
Lara S R Small, Marc Bruning, Andrew R Thomson, Aimee L Boyle, Roberta B Davies, Paul M G Curmi, Nancy R Forde, Heiner Linke, Derek N Woolfson, Elizabeth H C Bromley
Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed...
March 14, 2017: ACS Synthetic Biology
Gao-Yi Tan, Kunhua Deng, Xinhua Liu, Hui Tao, Yingying Chang, Jia Chen, Kai Chen, Zhi Sheng, Zixin Deng, Tiangang Liu
With the advent of the genomics era, heterologous gene expression has been used extensively as a means of accessing natural products (NPs) from environmental DNA samples. However, the heterologous production of NPs often has very low efficiency or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in native producers, especially in the cases of genome mining, it is also difficult to rationally and systematically engineer synthetic pathways to improved NPs biosynthetic efficiency...
March 13, 2017: ACS Synthetic Biology
Stephanie Tzouanas Schmidt, Stephanie M Zimmerman, Jianbin Wang, Stuart K Kim, Stephen R Quake
Lineage tracing by the determination and mapping of progeny arising from single cells is an important approach enabling the elucidation of mechanisms underlying diverse biological processes ranging from development to disease. We developed a dynamic sequence-based barcode system for synthetic lineage tracing and have demonstrated its performance in C. elegans, a model organism whose lineage tree is well established. The strategy we use creates lineage trees based upon the introduction of synthetically controlled mutations into cells and the propagation of these mutations to daughter cells at each cell division...
March 10, 2017: ACS Synthetic Biology
Orr Schlesinger, Yonatan Chemla, Mathias Heltberg, Eden Ozer, Ryan Marshall, Vincent Noireaux, Mogens Høgh Jensen, Lital Alfonta
Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression...
March 9, 2017: ACS Synthetic Biology
William Henry Bothfeld, Grace Kapov, Keith Tyo
Many biosynthetic strategies are coupled to growth, which is inherently limited, as (1) excess feedstock (e.g., sugar) may be converted to biomass, not product, (2) essential genes must be maintained and (3) growth toxicity must be managed. A decoupled growth and production phase strategy could avoid these issues. We have developed a toggle switch that uses glucose sensing to enable this two-phase strategy. Temporary glucose starvation precisely and autonomously activates product expression in rich or minimal media, obviating the requirement for expensive inducers...
March 8, 2017: ACS Synthetic Biology
Zhanar Abil, Laura F Gumy, Huimin Zhao, Casper C Hoogenraad
Localization of mRNA is important in a number of cellular processes such as embryogenesis, cellular motility, polarity, and a variety of neurological processes. A synthetic device that controls cellular mRNA localization would facilitate investigations on the significance of mRNA localization in cellular function and allow an additional level of controlling gene expression. In this work, we developed the PUF (Pumilio and FBF homology domain)-assisted localization of RNA (PULR) system, which utilizes a eukaryotic cell's cytoskeletal transport machinery to reposition mRNA within a cell...
March 8, 2017: ACS Synthetic Biology
Charlie Gilbert, Mark Howarth, Colin R Harwood, Tom Ellis
The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis...
March 7, 2017: ACS Synthetic Biology
Robert Sidney Cox, James Alastair McLaughlin, Raik Grünberg, Jacob Beal, Anil Wipat, Herbert M Sauro
As protein engineering becomes more sophisticated, practitioners increasingly need to share diagrams for communicating protein designs. To this end, we present a draft visual language, Protein Language, that describes the high-level architecture of an engineered protein with easy-to-draw glyphs, intended to be compatible with other biological diagram languages such as SBOL Visual and SBGN. Protein Language consists of glyphs for representing important features (e.g., globular domains, recognition and localization sequences, sites of covalent modification, cleavage and catalysis), rules for composing these glyphs to represent complex architectures, and rules constraining the scaling and styling of diagrams...
March 7, 2017: ACS Synthetic Biology
Shenghu Zhou, Renpeng Ding, Jian Chen, Guocheng Du, Hua-Zhong Li, Jingwen Zhou
Promoter is one of the most important and basic tools to achieve diverse of synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters have made implementing complicated strategies difficult. In this study, 104 native promoter-5'-UTR complexes (PUTR) from E...
March 2, 2017: ACS Synthetic Biology
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