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Scooped by Gerd Moe-Behrens
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CRISPR-SKIP: programmable gene splicing with single base editors

CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes through targeted double-strand breaks in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for off-target mutations, technologies capable of introducing targeted changes with increased precision, such as single-base editors, are preferred. We present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.
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Models give synthetic biologists a head start: Scientists build math tools to predict genetic circuits' performance

Models give synthetic biologists a head start: Scientists build math tools to predict genetic circuits' performance | SynBioFromLeukipposInstitute | Scoop.it
Researchers have developed mathematical models to predict the performance of multi-input synthetic biological circuits that can be used to engineer bacteria and other organisms to regulate cellular systems or perform functions they wouldn't in nature. Applications include biological sensing, chemical production and therapeutics such as probiotics to alter gut bacteria.
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Establishing a high-yielding cell-free protein synthesis platform derived from Vibrio natriegens

A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries (e.g., promoters, ribosome binding sites) in non-model organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 +/- 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E. coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax™ Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V. natriegens-derived CFPS platform is versatile, and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.
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CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window

CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window | SynBioFromLeukipposInstitute | Scoop.it
The capacity to diversify genetic codes advances our ability to understand and engineer biological systems1,2. A method for continuously diversifying user-defined regions of a genome would enable forward genetic approaches in systems that are not amenable to efficient homology-directed oligonucleotide integration. It would also facilitate the rapid evolution of biotechnologically useful phenotypes through accelerated and parallelized rounds of mutagenesis and selection, as well as cell-lineage tracking through barcode mutagenesis. Here we present EvolvR, a system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. This is achieved by directly generating mutations using engineered DNA polymerases targeted to loci via CRISPR-guided nickases. We identified nickase and polymerase variants that offer a range of targeted mutation rates that are up to 7,770,000-fold greater than rates seen in wild-type cells, and editing windows with lengths of up to 350 nucleotides. We used EvolvR to identify novel ribosomal mutations that confer resistance to the antibiotic spectinomycin. Our results demonstrate that CRISPR-guided DNA polymerases enable multiplexed and continuous diversification of user-defined genomic loci, which will be useful for a broad range of basic and biotechnological applications.
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CRISPR ‘barcodes’ map mammalian development in exquisite detail

CRISPR ‘barcodes’ map mammalian development in exquisite detail | SynBioFromLeukipposInstitute | Scoop.it
For the first time, scientists have wielded CRISPR to track a mammal’s development from a single egg into an embryo with millions of cells. The technological feat brings biologists a step closer to being able to trace the history of every one of the billions of cells in complex animals such as mice — offering an unprecedented window into development and disease. The work was published1 on 8 August in Science.

“This has been the holy grail for a while,” says Aaron McKenna, a geneticist at the University of Washington in Seattle, who was part of a previous effort that used CRISPR to study zebrafish development2. “It was great to see this paper come out.”

Over the years, biologists have used a variety of methods to track an organism’s development cell by cell, such as labelling them with dyes. But these tools are unable to follow cells through many divisions, let alone over an organism’s entire life. In the past two years, however, CRISPR–Cas9 genome editing has emerged as a potent tool for monitoring development in exquisite detail.

In zebrafish, for example, researchers have engineered special genetic sequences into the genome that act like recording tape2. CRISPR leaves its mark on these sequences by adding or deleting DNA, giving each cell a unique genetic barcode. These edits accumulate as the cells divide. By reading off the barcodes, scientists can reconstruct a cellular family tree, or lineage, showing how cells relate to each other.

Level up
Mammals such as mice have vastly more cells than do zebrafish. To track their development using CRISPR, a team led by Reza Kalhor, a molecular biologist at Harvard Medical School in Boston, Massachusetts, bred a line of mice that contained 60 of these barcoding sites scattered throughout their genomes — enough, in theory, to give a unique tag to each of an adult mouse’s 10 billion cells.

When the researchers looked at the pattern of mutations that accumulated in the barcodes of 12-day-old mouse embryos, they were able to trace the histories of cells in each embryo’s primitive heart and limbs, as well as its placenta.

The team also showed how barcoding can help to answer open questions about a mammal’s development. By examining brain tissue from embryos, they found that the barcode patterns were more similar between equivalent regions of the left and right side of the brain — indicating they had formed from recent cell divisions — than between cells from different regions of the same side. This pattern suggests that the axis that runs from front to back of the brain is formed before the one that runs from left to right — a timeline that neuroscientists have struggled to pin down with existing tools.

Jan Philipp Junker, a systems biologist at the Max Delbrück Center for Molecular Medicine in Berlin, says the study is an “important development”. He adds, however, that the way in which the authors have read out the barcodes — by looking at a collection of cells in a tissue sample rather than examining individual cells — currently prevents them from tracing out the cells’ lineage in fine detail, with the entire history of divisions plotted out. Kalhor says that the group is keen to explore methods for quickly reading out barcodes from individual cells in the future.

Tracing cell lineages in mice could be a useful tool for understanding the cellular basis for human disease, says McKenna. Cancer researchers, for example, could breed the barcode strain with their own mouse models of cancer to examine in detail how the disease disrupts cell division. “I think we’re a little way from that,” he says, “but this is a big step forward.”
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A synthetic biology based cell line engineering pipeline

An ideal host cell line for deriving cell lines of high recombinant protein production should be stable, predictable, and amenable to rapid cell engineering or other forms of phenotypical manipulation. In the past few years we have employed genomic information to identify “safe harbors” for exogenous gene integration in CHO cells, deployed systems modeling and optimization to design pathways and control strategies to modify important aspects of recombinant protein productivity, and established a synthetic biology approach to implement genetic changes, all with the goal of creating a pipeline to produce “designer” cell lines.
Chinese hamster ovary (CHO) cells are the preferred platform for protein production. However, the Chinese hamster genome is unstable in its ploidy, is subject to long and short deletions, duplications, and translocations. In addition, gene expression is subject to epigenetic changes including DNA methylation, histone modification and heterochromatin invasion, thus further complicating transgene expression for protein production in cell lines. With these issues in mind, we set out to engineer a CHO cell line highly amenable to stable protein production using a synthetic biology approach. We compiled karyotyping and chromosome number data of several CHO cell lines and sublines, identified genomic regions with high a frequency of gain and loss of copy number using comparative genome hybridization (CGH), and verified structural variants using sequencing data. We further used ATAC (Assay for Transposase-Accessible Chromatin) sequencing to study chromatin accessibility and epigenetic stability within the CHO genome. RNA-seq data from multiple cell lines were also used to identify regions with high transcriptional activity. Analysis of these data allowed the identification of several “safe harbor” loci that could be used for cell engineering.
Based on results of the data analysis and identification of “safe harbors”, we engineered an IgG producing cell line with a single copy of the product transgene as a template cell line. This product gene site is flanked by sequences for recombinase mediated cassette exchange, therefore allowing easy substitution of the IgG producing gene for an alternative product gene. Furthermore, a “landing pad” for multi-gene cassette insertion was integrated into the genome at an additional site. Together, these sites allowed engineering of new cell lines producing a fusion protein and Erythropoietin to be generated from the template cell line. To enable rapid assembly of product transgenes and genetic elements for engineering cell attributes into multi-gene cassettes, we adopted a golden-gate based synthetic biology approach. The assembly of genetic parts into multi-gene cassettes in a LEGO-like fashion allowed different combinations of genes under the control of various promoters to be generated quickly for introduction into the template cell line.
Using this engineered CHO cell line, we set out to study metabolism and product protein glycosylation for cell engineering. To guide the selection of genetic elements for cell engineering, we developed a multi-compartment kinetic model, as well as a flux model of energy metabolism and glycosylation. The transcriptome meta-data was used extensively to identify genes and isoforms expressed in the cell line and to estimate the enzyme levels in the model. The flux model was used to identify and the LEGO-like platform was used to implement the genetic changes that can alter the glycosylation pattern of the IgG produced by the template cell line. Concurrently we employed a systems optimization approach to identify the genetic alterations in the metabolic pathway to guide cell metabolism toward a favorable state. The model prediction is being implemented experimentally using the synthetic biology approach.
In conclusion, we have illustrated a pipeline of rational cell line engineering that integrates genomic science, systems engineering and synthetic biology approaches. The promise, the technical challenges and possible limitations will be discussed in this presentation.
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Creating a functional single-chromosome yeast

Creating a functional single-chromosome yeast | SynBioFromLeukipposInstitute | Scoop.it
Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.
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CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway

CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway | SynBioFromLeukipposInstitute | Scoop.it
CRISPR–Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.
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Designing with living systems in the synthetic yeast project

Synthetic biology is challenged by the complexity and the unpredictability of living systems. While one response to this complexity involves simplifying cells to create more fully specified systems, another approach utilizes directed evolution, releasing some control and using unpredictable change to achieve design goals. Here we discuss SCRaMbLE, employed in the synthetic yeast project, as an example of synthetic biology design through working with living systems. SCRaMbLE is a designed tool without being a design tool, harnessing the activities of the yeast rather than relying entirely on scientists’ deliberate choices. We suggest that directed evolution at the level of the whole organism allows scientists and microorganisms to “collaborate” to achieve design goals, suggesting new directions for synthetic biology.
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Bioengineering Strategies for Protein-Based Nanoparticles. 

In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP's intrinsic properties and to impart them with novel functionalities.
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Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells

Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells | SynBioFromLeukipposInstitute | Scoop.it
Hemoglobin in red blood cells (RBCs) carries oxygen to the tissues. Sickle cell disease is an inherited condition that involves abnormal hemoglobin. Current treatments entail modulating the level of fetal hemoglobin expression. Grevet et al. performed a CRISPR-Cas9 screen for regulators of fetal hemoglobin in RBCs and identified heme-regulated eIF2α kinase (HRI). Depleting the kinase in RBCs led to an increase in fetal hemoglobin levels and reduced sickling of cultured human RBCs. Thus, HRI may be a therapeutic target for sickle cell disease and other hemoglobin disorders.
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Protobiology

Protobiology | SynBioFromLeukipposInstitute | Scoop.it
Building semi-synthetic minimal cells We are combining top-down and bottom-up approaches to synthetic biology; using tools of protein engineering and molecular biology, together with novel synthetic cell technologies, to understand and modulate biological processes. Synthetic minimal cells can help us studying basic properties of life, understand the origin of life on Earth, and give guidance looking for life elsewhere in the Universe. We aim at building models for studying diseases, for creating novel therapies and for making custom biofactories. We apply engineering principles to biology: we build life, or grow machines. This talk gives broad overview of our lab's work. The top-down approach involves building on the biological ensemble, modifying existing cellular pathways to explore and control biology. The bottom-up approach involves building chemical systems capable of mimicking cellular processes - the synthetic minimal cells, that mimic properties of live cells. Those synthetic cells allow us studying and engineering complex biological pathways, building new biological functionalities, make better drugs and novel therapy strategies, and tracing the evolutionary history of life in model protocells. back to top bottom-up synthetic biology Building artificial chemical systems mimicking cells We have developed the first known system capable of non-enzymatic RNA synthesis inside model protocells. In this work, we have shown how citric acid can stabilize fatty acid liposomes in presence of divalent cations, allowing for non-enzymatic template director RNA primer extension inside the liposomes. This discovery bridges the RNA-world hypothesis or earliest life having RNA-based metabolism with the origin of compartmentalization based on fatty acid liposomes. (Adamala and Szostak, Science 2013) We also discovered a chemically-driven replication mechanism for model protocells (Zhu, Adamala, Zhang and Szostak, PNAS 2012). Together, those projects allow for drawing a plausible, complete protocell cycle: inside the protocells, RNA is replicated by template-directed non-enzymatic synthesis, and the protocells grow into filamentous threads by absorbing fatty acid molecules from micelles and divide in response to shear stress. RNA replication inside protocellsdivision via threadsRNA is encapsulated in protocellsactivated nucleotides enter via semi-permeable bilayerRNA template is copiedSource: Adamala and Szostak, Science 2013 As part of the same overarching goal of building a chemical system capable of Darwinian evolution, we have shown that an encapsulated small peptide catalyst can impact the fitness of model protocells. I have studies catalysis of Ser-His and other di- and tri- amino acid catalytic peptides. We have shown that the minimal serine protease analogue, Ser-His, can catalyze formation of peptide nucleic acids (Gorlero et.al FEBS Letters 2009). We have also demonstrated how the same dipeptide catalyst's activity can be enhanced by the presence of fatty acid protocell vesicles, and in turn the product of the reaction catalysed by said dipeptide allows the protocell to enhance uptake of membrane building blocks, resulting in protocell growth. This couples the activity of encapsulated catalyst with the fitness of the protocell, in a model system exploring the origin of Darwinian selection. (Adamala and Szostak, Nature Chemistry 2013). Ser-His catalysed reactionSource: Adamala and Szostak, Nature Chemistry 2013 Work done with Pier Luigi Luisi from University Roma Tre and Jack Szostak from Harvard University. Our most significant publications in that area: Non-enzymatic template-directed RNA synthesis inside model protocells; K. Adamala and J.W. Szostak, Science 342 (2013) 1098 - 1100; local copy pdf publisher website link Competition between model protocells driven by an encapsulated catalyst; K. Adamala and J.W. Szostak, Nature Chemistry 5 (2013) 495 - 501; local copy pdf publisher website link Engineering genetic circuit interactions within and between synthetic minimal cells; Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, Katriona R. Guthrie-Honea, Edward S. Boyden; Nature Chemistry, 9, 431-439, 2017, doi:10.1038/nchem.2644; *equal contribution publisher website link local copy pdf Collaboration between primitive cell membranes and soluble catalysts; K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, doi:10.1038/ncomms11041; *equal contribution local copy pdf publisher website link Full list of peer reviewed publications: protobiology.org/publications back to top Broader impact TED talk on synthetic life Kate presented concept of building synthetic minimal cells, with its biotechnological, biomedical and basic science implications, in a TEDx talk . Protocells in the news The effort towards building life in the lab and elucidating the origin and earliest evolution of life has always been receiving interest from broad audience. STAT magazine published nice overview of efforts to build artificial life, From chemicals to life: Scientists try to build cells from scratch. Some examples of the astrobiology focus: a review detailing our work on RNA replication in protocells: Angewandte Chemie International Edtion Citric Acid and the RNA World 2014, 53, 5245 - 5247, external link (local copy pdf) Our work on protocells have been featured in science news outlets and editorials, including: Science Focus: Robert Service, The Life Force, external link (local copy pdf) BioTechniques: How Cellular Life Evolved 06 Jan 2014 external link (local copy pdf) Science News: To cook up life, just add citrate 185(1):15 external link (local copy pdf) Science Daily: Researchers find missing component in effort to create primitive, synthetic cells, external link (local copy pdf) Chemical & Engineering News: Lab-Made Protocells Show Hints Of Evolution, 91(21), 2013 The Panda's Thumb: New Szostak protocell is closest approximation to origin of life and Darwinian evolution so far, external link (local copy pdf) back to top top-down synthetic biology Engineering generalized RNA-protein interactions: a toolbox for regulation and readout of gene expression We developed and validated protein architecture which binds to single stranded RNA. Using this protein technology, we are developing tools for visualization and quantification of levels of expression of genes of interest. This tool will work by following the reconstitution of a protein probe upon interaction of sequence-specific RNA binding proteins with the mRNA of the gene of interest. We are also aiming to edit the transcriptome of the gene of interest, selectively decreasing the level of expression of one splice variant (as opposed to cutting the DNA of the gene, which targets all splice variants indiscriminately). This technology could potentially help in studying non-ER translation events, elucidating mechanisms of synaptic plasticity, as well as studying healthy and diseased translational profiles of genes, e.g., those involved in oncogenesis and other disease processes. The ability to monitor and perturb RNA in living neurons - which would open up the investigation of many processes that contribute to development, plasticity, and disease progression - would benefit greatly from a method of systematically targeting unmodified RNA sequences for observation and control. The current ssRNA targeting methods are based on the RNA binding protein aptamers, like MS2 or PP7; this require the introduction of aptamer binding sites into the RNA. This work shows that it is possible to develop a ssRNA binding protein that can be engineered to target arbitrary sequences of variable length, thus eliminating the need to engineer the target sites into the RNA of interest. Popular science description: news release, local copy pdf. This work was done with Ed Boyden
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Synthetic biology-inspired design of signal-amplifying materials systems

Synthetic biology-inspired design of signal-amplifying materials systems | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology applies engineering concepts to build cells that perceive and process information. Examples include cells engineered to perform basic digital or analog computation. These circuits serve as basis for the construction of complex integrated cellular networks that offer manifold applications in fundamental and applied research. Here, we introduce the concept of using design approaches and molecular tools applied in synthetic biology for the construction of interconnected biohybrid materials systems with information processing functionality. We validate this concept by modularly assembling protein and polymer building blocks to generate stimulus-responsive materials. Guided by a quantitative mathematical model, we next interconnect these materials into a materials system that acts as both a signal detector and as an amplifier based on a built-in positive feedback loop. The functionality and versatility of this materials system is demonstrated by the detection of enzymatic activities and drugs. The modular design concept presented here thus represents a blueprint for integrating synthetic biology-inspired information-processing circuits into polymer materials. As integrated sensors and actuators, the resulting smart materials systems could provide novel solutions with broad perspectives in research and development.
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Math cuts trial and error in building biological circuits

Math cuts trial and error in building biological circuits | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biologists have the tools to build complex, computer-like DNA circuits that sense or trigger activities in cells. And thanks to new research, they now have a way to test those circuits in advance.

Researchers developed models to predict the output of custom-built genetic circuits that, for example, scientists can prompt to start or stop the production of proteins.

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Frontiers | “Do It Yourself” Microbial Cultivation Techniques for Synthetic and Systems Biology: Cheap, Fun, and Flexible

Frontiers | “Do It Yourself” Microbial Cultivation Techniques for Synthetic and Systems Biology: Cheap, Fun, and Flexible | SynBioFromLeukipposInstitute | Scoop.it
With the emergence of inexpensive 3D printing technology, open-source platforms for electronic prototyping and single-board computers, “Do it Yourself” (DIY) approaches to the cultivation of microbial cultures are becoming more feasible, user-friendly, and thus wider spread. In this perspective, we survey some of these approaches, as well as add-on solutions to commercial instruments for synthetic and system biology applications. We discuss different cultivation designs, including capabilities and limitations. Our intention is to encourage the reader to consider the DIY solutions. Overall, custom cultivation devices offer controlled growth environments with in-line monitoring of, for example, optical density, fluorescence, pH, and dissolved oxygen, all at affordable prices. Moreover, they offer a great degree of flexibility for different applications and requirements and are fun to design and construct. We include several illustrative examples, such as gaining optogenetic control and adaptive laboratory evolution experiments.

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Asimov - Bring Computation to Life

Asimov - Bring Computation to Life | SynBioFromLeukipposInstitute | Scoop.it
Asimov is a startup that programs living cells with genetic circuits. We
partner with companies to create previously impossible biotechnologies. Our
engineering platform combines computer-aided design, artificial
intelligence, and synthetic biology.
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Did CRISPR really fix a genetic mutation in these human embryos?

Did CRISPR really fix a genetic mutation in these human embryos? | SynBioFromLeukipposInstitute | Scoop.it
Researchers provide more evidence for their landmark claim that gene editing rid embryos of a disease mutation — but scientists are still arguing over the results.
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Synthesis at the interface of virology and genetic code expansion

How a virus efficiently invades its host cell and masterfully engineers its properties provides valuable lessons and resources for the emerging discipline of synthetic biology, which seeks to create engineered biological systems with novel functions. Recently, the toolbox of synthetic biology has also been enriched by the genetic code expansion technology, which has provided access to a large assortment of unnatural amino acids with novel chemical functionalities that can be site-specifically incorporated into proteins in living cells. The synergistic interplay of these two disciplines holds much promise to advance their individual progress, while creating new paradigms for synthetic biology. In this review we seek to provide an account of the recent advances at the interface of these two research areas.
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BioBits™ Explorer: A modular synthetic biology education kit

BioBits™ Explorer: A modular synthetic biology education kit | SynBioFromLeukipposInstitute | Scoop.it
Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.
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The role of the encapsulated cargo in microcompartment assembly

The role of the encapsulated cargo in microcompartment assembly | SynBioFromLeukipposInstitute | Scoop.it
Bacterial microcompartments are large, roughly icosahedral shells that assemble around enzymes and reactants involved in certain metabolic pathways in bacteria. Motivated by microcompartment assembly, we use coarse-grained computational and theoretical modeling to study the factors that control the size and morphology of a protein shell assembling around hundreds to thousands of molecules. We perform dynamical simulations of shell assembly in the presence and absence of cargo over a range of interaction strengths, subunit and cargo stoichiometries, and the shell spontaneous curvature. Depending on these parameters, we find that the presence of a cargo can either increase or decrease the size of a shell relative to its intrinsic spontaneous curvature, as seen in recent experiments. These features are controlled by a balance of kinetic and thermodynamic effects, and the shell size is assembly pathway dependent. We discuss implications of these results for synthetic biology efforts to target new enzymes to microcompartment interiors.
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Blueprints for Biosensors: Design, Limitations, and Applications

Biosensors are enabling major advances in the field of analytics that are both facilitating and being facilitated by advances in synthetic biology. The ability of biosensors to rapidly and specifically detect a wide range of molecules makes them highly relevant to a range of industrial, medical, ecological, and scientific applications. Approaches to biosensor design are as diverse as their applications, with major biosensor classes including nucleic acids, proteins, and transcription factors. Each of these biosensor types has advantages and limitations based on the intended application, and the parameters that are required for optimal performance. Specifically, the choice of biosensor design must consider factors such as the ligand specificity, sensitivity, dynamic range, functional range, mode of output, time of activation, ease of use, and ease of engineering. This review discusses the rationale for designing the major classes of biosensor in the context of their limitations and assesses their suitability to different areas of biotechnological application.
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Cellular Computing and Synthetic Biology

Cellular Computing and Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is an emerging, rapidly growing research field in which engineering principles are applied to natural, living systems. A major goal of synthetic biology is to harness the inherent “biological nanotechnology” of living cells for a number of applications, including computation, production, and diagnosis. In its infancy, synthetic biology was mainly concerned with the construction of small-scale, proof-of-principle computational devices (cellular computing), along the lines of simple logic gates and circuits, but the state-of-the-art now uses multicellular complexes and engineered cell-cell communication. From its practical origins around the turn of the century, the field has grown into a global scientific market predicted to be worth tens of billions of dollars by 2020. Anticipated applications include tissue engineering, environmental remediation, in situ disease detection and treatment, and even the development of the first fully-synthetic organism. In this chapter we review the timeline of synthetic biology, describe its alignment with unconventional computation, and, drawing on quotations from leading researchers in the field, describe its main challenges and opportunities.
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A Synthetic Bacterial - Adhesion Toolbox for Programming Multicellular Morphologies and Patterns

A Synthetic Bacterial - Adhesion Toolbox for Programming Multicellular Morphologies and Patterns | SynBioFromLeukipposInstitute | Scoop.it
Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.
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Construction of integrated gene logic-chip

Construction of integrated gene logic-chip | SynBioFromLeukipposInstitute | Scoop.it
In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.
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