scholarly journals A GoldenBraid cloning system for synthetic biology in social amoebae

2020 ◽  
Vol 48 (8) ◽  
pp. 4139-4146 ◽  
Author(s):  
Peter Kundert ◽  
Alejandro Sarrion-Perdigones ◽  
Yezabel Gonzalez ◽  
Mariko Katoh-Kurasawa ◽  
Shigenori Hirose ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Proof Of Concept ◽  
Host Interactions ◽  
Fluorescent Reporters ◽  
Cloning System ◽  
Genetic Elements ◽  
Social Amoebae

Abstract GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria–host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.

scholarly journals A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

2020 ◽  
Author(s):  
Simon J Moore ◽  
Hung-En Lai ◽  
Soo-Mei Chee ◽  
Ming Toh ◽  
Seth Coode ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Gene Clusters ◽  
Test Tube ◽  
High Yield ◽  
Proof Of Concept ◽  
Biosynthetic Gene Clusters ◽  
Reaction Conditions ◽  
One Pot ◽  
Free System ◽  
High Level

AbstractProkaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single ‘one-pot’ reaction.

scholarly journals The Software Crisis of Synthetic Biology

2016 ◽  
Author(s):  
Sergi Valverde ◽  
Manuel Porcar ◽  
Juli Pereto ◽  
Ricard V. Sole
Keyword(s):  
Complex System ◽  
Software Engineering ◽  
Synthetic Biology ◽  
Large Scale ◽  
Computer Models ◽  
Industrial Engineering ◽  
Proof Of Concept ◽  
Software Repository ◽  
Software Crisis ◽  
Standard Components

In fifteen years, Synthetic Biology (SB) has moved from proof-of-concept designs to several flagship achievements. Standardisation efforts are still under way, basic engineering concepts such as modularity and orthogonality are still controversial in biology, and making predictions from computer models is still unreliable. A deep characterization in the pattern of re-use of biological blocks in SB has not been attempted to date. We have compared the topological organisation of two different technological networks, one associated to a standard, large-scale software repository and the second provided by the Registry of Standard Biological Parts (RSBP). Our results strongly suggest that software engineering, and not industrial engineering, is the closest complex system to SB. In both cases, combining standard or quasi-standard components assembly with tinkering may not be at odds with success.

scholarly journals GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Roland Prielhofer ◽  
Juan J. Barrero ◽  
Stefanie Steuer ◽  
Thomas Gassler ◽  
Richard Zahrl ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Pichia Pastoris ◽  
Golden Gate ◽  
Yeast Pichia Pastoris ◽  
Cloning System ◽  
Modular Cloning

Carbohydrate microarrays: key developments in glycobiology

2009 ◽  
Vol 390 (7) ◽  
Author(s):  
Yan Liu ◽  
Angelina S. Palma ◽  
Ten Feizi
Keyword(s):  
Protein Interactions ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Biological Information ◽  
Biological Processes ◽  
Proof Of Concept ◽  
Biomedical Sciences ◽  
Host Interactions ◽  
New Development ◽  
Medical Importance

Abstract Carbohydrate chains of glycoproteins, glycolipids, proteoglycans, and polysaccharides mediate processes of biological and medical importance through their interactions with complementary proteins. The unraveling of these interactions is therefore a priority in biomedical sciences. Carbohydrate microarray technology is a new development at the frontier of glycomics that is revolutionizing the study of carbohydrate-protein interactions and the elucidation of their specificities in endogenous biological processes, microbe-host interactions, and immune defense mechanisms. In this review, we briefly refer to the principles of numerous platforms since the introduction of carbohydrate microarrays in 2002, and we highlight platforms that are beyond proof-of-concept and have provided new biological information.

scholarly journals STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

2021 ◽  
Author(s):  
Catarina Grandela ◽  
Albert Blanch-Asensio ◽  
Karina O Brandao ◽  
Tessa de Korte ◽  
Loukia Yiangou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Synthetic Biology ◽  
Homologous Recombination ◽  
Pluripotent Stem Cells ◽  
Mammalian Cells ◽  
Site Specific ◽  
Fluorescent Reporters ◽  
Contraction Coupling ◽  
Induced Pluripotent ◽  
Multiple Variants

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) by: (i) inserting various combinations of fluorescent reporters into hiPSCs to assess excitation-contraction coupling cascade in derivative cardiomyocytes, and; (ii) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorder into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically-matched panels of hiPSC lines efficiently and cost-effectively.

scholarly journals Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

2021 ◽  
Author(s):  
Vivianne J Goosens ◽  
Kenneth T Walker ◽  
Silvia M Aragon ◽  
Amritpal Singh ◽  
Vivek R Senthivel ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Protein Secretion ◽  
Functional Expression ◽  
Dna Assembly ◽  
Cloning System ◽  
Pure Cellulose ◽  
Modular Cloning ◽  
High Yields ◽  
Short Time ◽  
Cellulose Materials

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

A Pragmatist Account of Functions in Synthetic Biology

2019 ◽  
Vol 96 (1) ◽  
pp. 171-186
Author(s):  
Johannes Achatz
Keyword(s):  
Synthetic Biology ◽  
Function Theory ◽  
Science And Technology ◽  
Proof Of Concept ◽  
Biological Functions ◽  
Advantages And Disadvantages ◽  
Technical Functions ◽  
Biological Functionality ◽  
Biology Research ◽  
Normative Account

In fields where science and technology overlap, so do different function-ascriptions. The entities of Synthetic Biology research are a case in point, where organisms with biological functionality are altered to perform technical functions. A function theory for SynBiofacts has to address artifactual as well as biological functions of one and the same entity. Further demands on a function theory for Synthetic Biology emerge from methods of SynBiofact creation called kludging and the use-scenarios of SynBiofacts in proof-of-concept research and BioArt. After discussing intentional accounts of technical functions, etiological function theory, contextual causation and contextual selection, a pluralist and weakly normative account of function will be outlined, that, or so it will be argued, can indeed cover all function ascriptions in Synthetic Biology. Lastly, some of the advantages and disadvantages of taking this stance will be outlined.

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