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MANIPULATING MICROBIAL METABOLISM

MANIPULATING MICROBIAL METABOLISM | SynBioFromLeukipposInstitute | Scoop.it
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by
Eva J. Gordon

"Reactive oxygen species (ROS), chemically reactive substances such as hydrogen peroxide (H2O2) and superoxide anion radical (O2−), are normal byproducts of oxygen metabolism in organisms ranging from bacteria to humans. However, their high reactivity contributes to their destructive side, which manifests as damage to various biomolecules including DNA, RNA, proteins, and lipids. The destructive properties of ROS in microbes can be exploited to help kill pathogenic varieties, a skill that is desperately needed to combat the growing number of antibiotic-resistant bacteria that have emerged across the globe. However, the networks responsible for ROS production in bacteria are not well delineated. Now, Brynildsen et al. (Nat. Biotechnol. 2013, 31, 160−165) use computational metabolic modeling to guide strategies for manipulating ROS production in bacteria for the purpose of enhancing the organism’s susceptibility to toxic agents...."

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CLOT Magazine | Ginkgo Bioworks

CLOT Magazine | Ginkgo Bioworks | SynBioFromLeukipposInstitute | Scoop.it
@Ginkgoo,a #biotech company designing&engineering custom organisms as biological solutions https://t.co/uSYmXAcnwF #biotechnology #science https://t.co/77ycXu9bMs
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Engineering genetic circuit interactions within and between synthetic minimal cells

Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.
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Sensitive CRISPR diagnostics using RNA targeting CRISPR enzyme | PLOS Synthetic Biology Community

Sensitive CRISPR diagnostics using RNA targeting CRISPR enzyme | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
I was recently involved in a collaboration between the Zhang and Collins labs at MIT to use the RNA-targeting CRISPR protein Cas13a/C2c2 to detect either DNA or RNA from pathogens. By combining the use of Cas13a/C2c2 as a detector with isothermal amplification of the DNA or RNA targets, we were able to get down to attomolar detection. You can read the full paper over at Science but here I can give some of my own experience with and views on the platform we’re calling SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).

To be clear, I’m the 5th author on this paper and definitely agree that the four people ahead of me did more of the work. I am not the expert on Cas13a/C2c2. However, I did help some to develop SHERLOCK as a diagnostic system and use it enough to get a handle for how it works with different targets. I also tried it with another diagnostic project and have found it easy to work with.

How it works

Cas13a/C2c2 has two RNA cutting abilities. The first is that it cuts the RNA that you target with theCRISPR guide RNA (crRNA) much like Cas9 cuts DNA that you directly target. The other RNA cutting is more broad and is activated after finding Cas13a/C2c2 finds its target RNA. This broad RNA cutting activity acts like an RNase and will cut many RNAs present in the reaction (or in the cell). In a 2016 Nature paper, the Doudna lab showed that the RNase activity of Cas13a/C2c2 could be used to detect picomolar levels of RNA. They found that Cas13a/C2c2 was able to do at least 104 turnovers per target RNA recognized. That catalytic activity of Cas13a/C2c2 gives a strong output signal for even s small input of target RNA.

So Cas13a/C2c2 can be turned into a diagnostic using just a crRNA and a fluorescent RNA probe. After the Cas13a/C2c2 finds its target it starts cutting other RNAs, including the probe, and separates a fluorophore from its quencher. It’s that separation of fluorophore and quencher that gives the fluorescent signal.

To boost the natural sensitivity of Cas13a/C2c2 as a diagnostic, we paired it with isothermal amplification of DNA or RNA. Isothermal amplification methods amplify nucleic acids similar to polymerase chain reaction (PCR) but instead use enzyme mixes that can do the job at a single temperature. SHERLOCK makes use of recombinase polymerase amplification (RPA) that can work between 37-42˚C. This allows both amplification reactions and Cas13a/C2c2 reactions at 37˚C and means that there is no need for expensive machinery to precisely cycle temperatures like PCR.

Advantages

The level of sensitivity we got certainly jumps off of the page. As mentioned in the last section, Cas13a/C2c2 is itself quite sensitive to RNA molecules and its collateral RNase activity can cut many probe RNAs. It binds to its target RNA and then quickly generates signal through its general RNA cutting activity. As a detector it’s ~1000 times more sensitive than another detector, the RNA toehold switch, that we’ve used in the Collins lab to things like detect Zika.

Similar to the paper-based Zika detection, SHERLOCK was able to be freeze-dried for room-temperature storage, used with minimal hardware, and rapidly reprogrammed rapidly to target almost any sequence. But in addition to the sensitivity advantage, SHERLOCK was able to detect single base mutations. As many important mutations in humans or the pathogens that infect humans are only single bases, the ability to distinguish those small changes would be a major achievement for a cheap diagnostic. Some screening has to be done to find crRNAs that work best for a given mutation, but in general a few variants should be enough.

Potential limitations

This is still early days for Cas13a/C2c2 based CRISPR diagnostics, so there will be more challenges to be addressed in academic labs and in a company setting. While we showed freeze-drying on glass fiber paper and adding RNase inhibitor worked without producing much background signal, samples that contain many RNases could create false positives. A negative control that lacks Cas13a/C2c2or crRNA could inform you of the problem but the RNase containing sample likely couldn’t give you an accurate read of how much nucleic acid is actually present. At the lab bench, we didn’t have problems with background signal but working somewhere like a remote community health center would probably bring less controlled conditions. Rigorous tests will need to be done to make sure that the freeze-dried tests can last out in different conditions.

Other improvements could include a good way to change from a fluorescent output to a color change as the output. This would allow easy readout by eye like a pregnancy test and reduce equipment costs. A color readout can be done by anyone without risk of equipment malfunction. Reducing the equipment and technical skill needed for a diagnostic is key to how easily it can actually be deployed in areas that need it.

Future for CRISPR diagnostics

The variety of CRISPR proteins that target DNA or RNA and can be easily programmed to cut or bind to nearly any sequence. Cas13a/C2c2 is nice because it comes with a secondary activity (general RNA cutting) that can readily be turned into a fluorescent readout. However, CRISPR-Cas9 can also be used for diagnostics when cleverly combined with a way of detecting its targeted DNA cutting. Overall, CRISPR proteins are poised to get integrated into nucleic acid diagnostics as they provide programmable detection and more specificity than traditional nucleic acid amplification-based techniques.

 

See more coverage over at Science Magazine, MIT News, Washington Post, The Scientist, and STAT News.
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Directed evolution in synthetic biology: an interview with Professor Frances Arnold | PLOS Synthetic Biology Community

Directed evolution in synthetic biology: an interview with Professor Frances Arnold | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
At the beginning of my scientific career, I was captivated by the ability of organic chemists to synthesize molecules.  I soon realised, however, that the effort involved was often incredible, especially when you wanted to have control over the stereochemistry of complex molecules. Luckily for me, I also learned that there was another way to make these molecules, using Nature’s machinery. Nature has been synthesizing molecules and materials for billions of years, and Darwinian evolution has produced an immense array of beautiful biocatalysts (enzymes) that can assemble breathtaking structures. Scientists working in the fields of biocatalysis and synthetic biology exploit the power of these natural catalysts to find greener and easier routes for chemical synthesis. The problem is that, even though Nature has gifted us with many biocatalysts, they are not always suitable for exactly what we would like them to do. Hence, the importance of being able to engineer them, evolve their functions to match what we need.

If today we are able to ‘easily’ engineer enzymes in the laboratory, we owe this in large part to the work of Professor Frances Arnold: the mother of directed evolution. Prof. Arnold is an engineer and a biochemist, and a Dickinson Professor at the California Institute of Technology (Caltech). Author of over 200 peer-reviewed articles, she holds an impressive list of awards, and she recently was invited to Université de Montréal, where she presented two lectures under the prestigious Roger-Barré program.

I could not miss this unique opportunity to interview her for our blog. It is a pleasure to share with our synthetic biology community her inspiring insight about protein engineering in synthetic biology.
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Microsoft is buying another 10 million strands of DNA for storage research

Microsoft is buying another 10 million strands of DNA for storage research | SynBioFromLeukipposInstitute | Scoop.it
DNA storage could allow vast amounts of data to be stored for thousands of years.
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Designing uniquely addressable bio-orthogonal synthetic scaffolds for DNA and RNA origami

Nanotechnology and synthetic biology are rapidly converging, with DNA origami being one of the leading bridging technologies. DNA origami was shown to work well in a wide array of biotic environments. However, the large majority of extant DNA origami scaffolds utilize bacteriophages or plasmid sequences thus severely limiting its future applicability as bio-orthogonal nanotechnology platform. In this paper we present the design of biologically inert (i.e. "bio-orthogonal") origami scaffolds. The synthetic scaffolds have the additional advantage of being uniquely addressable (unlike biologically derived ones) and hence are better optimised for high-yield folding. We demonstrate our fully synthetic scaffold design with both DNA and RNA origamis and describe a protocol to produce these bio-orthogonal and uniquely addressable origami scaffolds.
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Exploring the potential of Saccharomyces cerevisiae for biopharmaceutical protein production

Production of recombinant proteins by yeast plays a vital role in the biopharmaceutical industry. It is therefore desirable to develop yeast platform strains for over-production of various biopharmaceutical proteins, but this requires fundamental knowledge of the cellular machinery, especially the protein secretory pathway. Integrated analyses of multi-omics datasets can provide comprehensive understanding of cellular function, and can enable systems biology-driven and mathematical model-guided strain engineering. Rational engineering and introduction of trackable genetic modifications using synthetic biology tools, coupled with high-throughput screening are, however, also efficient approaches to relieve bottlenecks hindering high-level protein production. Here we review advances in systems biology and metabolic engineering of yeast for improving recombinant protein production.
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A New Flavor of CRISPR Could Tackle Some of the Worst Genetic Diseases

A New Flavor of CRISPR Could Tackle Some of the Worst Genetic Diseases | SynBioFromLeukipposInstitute | Scoop.it
Most of the time, when people talk about the cutting edge gene editing technology CRISPR, they are actually talking about CRISPR-Cas9. CRISPR, you see, is just one half of the genome editing tool, the programming that instructs where a DNA edit will actually be made. The other part consists of proteins that actually do the cutting. And one particular protein, called Cas9, has long been the snipping tool of choice. But now, there’s a new protein on the block—and it may open the door to curing a devastating genetic disease.

Scientists have long suspected that another protein, Cpf1, might actually be superior to Cas9 because it is a smaller, simpler enzyme. And now, for the first time, scientists have successfully used it to edit human cells.

In a new paper out Wednesday in Science Advances, researchers from the University of Texas detail using CRISPR-Cpf1 to correct mutations associated with Duchenne muscular dystrophy, a disease that results in muscular degeneration. In human heart muscle cells, the researchers were able to correct key mutations and prevent disease progression. And in mice with the disease, they were able to reverse symptoms like inflammation.

The authors note that CRISPR-Cpf1 may actually be more successful at targeting Duchenne muscular dystrophy, by accessing mutation sites the more unwieldy Cas9 can’t access. Cpf1, they said, could be a powerful new tool in the CRISPR arsenal. There are many different mutations associated with human disease, and not every mutation is compatible with Cas9. But some of those, as in the case of Duchenne, might be compatible with Cpf1.

In the midst of an ongoing legal battle over who owns the rights to CRISPR-Cas9, the new study highlights the fact that pace at which science moves may make today’s legal skirmishes far less relevant before they are ever settled. In 2015, the Broad Institute first suggested that CRISPR-Cpf1 might be a superior system. There are also a slew of other CRISPR alternatives in the works. In May, Chinese researchers published a controversial paper on NgAgo, an entirely new system that they said can also be used to edit mammalian DNA. In June, researchers from several universities announced the discovery of C2c2, a CRISPR enzyme that targets RNA, rather than DNA. And just before Christmas, researchers at Berkeley announced the discovery of two new CRISPR/Cas systems, CRISPR-CasX and CRISPR-CasY.

Most importantly, though, the study paves the way for more research into how Cpf1 might be used to cure diseases and conditions that even CRISPR-Cas9 has so far not yet been able to tackle.
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Synthetic Biology: Building a custom eukaryotic genome de novo

Synthetic Biology: Building a custom eukaryotic genome de novo | SynBioFromLeukipposInstitute | Scoop.it
The Synthetic Yeast Project (Sc2.0) aims to create the first synthetic eukaryotic genome. It is based on synthesizing, from scratch, a reworked Saccharomyces cerevisiae genome that is optimized for genomic stability and includes various design features to make it an easily engineerable chassis for future applications.
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Synthetic biology and metabolic engineering approaches and its impact on non-conventional Yeast and Biofuel production

The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. One of the most promising alternatives to petroleum for the production of fuels is microbial production. Yeasts are highly efficient producer of bioethanol with several superior traits over bacterial counterparts. Tools of synthetic biology has revolutionised the field of microbial cell factories especially in the case of ethanol and fatty acid production. Era of yeast synthetic biology began with the well-studied industrial work horse Saccharomyces cerevisiae. Despite many highly beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, non-conventional yeast has for synthetic biology, it currently lags behind Sachharomyces cerevisiae in the number of synthetic networks that have been described. Currently synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris and Yarrowia lipolytica. Here we review basic synthetic biology tools that we can apply to non-conventional yeasts. Moreover we discuss how metabolic engineering and synthetic biology tools can be applied in nonconventional yeasts for improved biofuel production.
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An engineering paradigm in the biomedical sciences: Knowledge as epistemic tool

In order to deal with the complexity of biological systems and attempts to generate applicable results, current biomedical sciences are adopting concepts and methods from the engineering sciences. Philosophers of science have interpreted this as the emergence of an engineering paradigm, in particular in systems biology and synthetic biology. This article aims at the articulation of the supposed engineering paradigm by contrast with the physics paradigm that supported the rise of biochemistry and molecular biology. This articulation starts from Kuhn's notion of a disciplinary matrix, which indicates what constitutes a paradigm. It is argued that the core of the physics paradigm is its metaphysical and ontological presuppositions, whereas the core of the engineering paradigm is the epistemic aim of producing useful knowledge for solving problems external to the scientific practice. Therefore, the two paradigms involve distinct notions of knowledge. Whereas the physics paradigm entails a representational notion of knowledge, the engineering paradigm involves the notion of ‘knowledge as epistemic tool’.
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Scientists Get Closer to Building Artificial Life

Despite ethical and safety concerns, researchers are getting closer to building life from scratch. In fact, scientists are hoping to synthesize a huma
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CRISPR-Cas: Adapting to change

The arms race between prokaryotes and their perpetually evolving predators has fueled the evolution of a defense arsenal. The so-called CRISPR-Cas systems—clustered regularly interspaced short palindromic repeats and associated proteins—are adaptive immune defense systems found in bacteria and archaea. The recent exponential growth of research in the CRISPR field has led to the discovery of a diverse range of CRISPR-Cas systems and insight into their defense functions. These systems are divided into two major classes and six types. Each system consists of two components: a locus for memory storage (the CRISPR array) and cas genes that encode the machinery driving immunity. Information stored within CRISPR arrays is used to direct the sequence-specific destruction of invading genetic elements, including viruses and plasmids. As such, all CRISPR-Cas immune systems are reliant on the formation of CRISPR memories, known as spacers, to facilitate future defense. To form these memories, small fragments of invader nucleic acids are added as spacers to the CRISPR memory banks in a process termed CRISPR adaptation. The genetic basis of immunity means that CRISPR adaptation provides heritable benefits, an attribute that is unparalleled in eukaryotic immune systems. There is widespread evidence of highly active CRISPR adaptation in nature, and it is clear that these systems play important roles in shaping microbial evolution and global ecological networks.
ADVANCES
CRISPR adaptation requires several processes, including selection and processing of spacer precursors and their subsequent localization to, and integration into, the CRISPR loci. Although our understanding of all facets of the CRISPR adaptation pathway is not yet complete, considerable progress has been made in the past few years. At the heart of CRISPR adaptation is a protein complex, the Cas1-Cas2 “workhorse,” which catalyzes the addition of new spacers to CRISPR memory banks. A combination of functional assays and high-resolution structures of Cas1-Cas2 complexes has recently led to major advances. There is now a sound understanding of how foreign DNA is converted to prespacer substrates and captured by the Cas1-Cas2 complex. After this, Cas1-Cas2 locates the genomic CRISPR locus and docks in the appropriate position for insertion of the new spacer into the CRISPR array, while duplicating a CRISPR repeat. The cues directing the docking of substrate-laden Cas1-Cas2 differ between systems, with some relying on intrinsic sequence specificity and others assisted by host proteins.

Before integration, accurate processing of the spacer precursors is required to ensure that the new spacers are compatible with the protein machinery in order to elicit CRISPR-Cas defense. For a given CRISPR-Cas system, spacers must typically be of a certain length and be inserted into the CRISPR in a specific orientation. It is becoming increasingly apparent that Cas1-Cas2 complexes from diverse systems are capable of ensuring that these system-specific factors are met with high fidelity.

New findings also account for the ordering of stored memories: Typically, the insertion of new spacers is directed to one end of CRISPR arrays, and it has been shown that this enhances immunity against recently encountered invaders. The chronological ordering of new spacers has enabled insights into the temporal dynamics of interactions between hosts and invaders that are constantly changing. Some CRISPR-Cas systems use existing spacers to recognize previously encountered elements and promote the formation of new CRISPR memories, a process known as primed CRISPR adaptation. Viruses and plasmids that have escaped previous CRISPR-Cas defenses through genetic mutations trigger primed CRISPR adaptation. Several recent studies have revealed that primed CRISPR adaptation is also strongly promoted by recurrent invaders, even in the absence of escape mutations. This has led to previously separate paradigms of invader destruction and primed CRISPR adaptation beginning to converge into a unified model.
OUTLOOK
CRISPR adaptation is crucial for ensuring both population-level protection through spacer diversity and protection of the host through invader clearance. Although many studies have explored CRISPR adaptation in a broad range of host-specific and metagenomic contexts, much of the mechanistic detail has been gleaned from studying a relatively small subset of systems. Thus, despite the relative wealth of mechanistic information about CRISPR adaptation in a few specific types, work in other systems continues to reveal distinct modes of operation for spacer acquisition. Therefore, studies of CRISPR adaptation in alternative systems are necessary to determine which processes are conserved and which are system-specific. An important remaining question is why the enhanced primed CRISPR adaptation commonly found in type I systems has not yet been observed in other types. Do other systems possess analogous mechanisms that have yet to be discovered, or does the absence of priming in these systems explain the prevalence of type I systems in nature? Future expansion of our understanding of how CRISPR adaptation is carried out in the diverse repertoire of CRISPR-Cas systems is vital for maximizing the potential for repurposing the spacer acquisition machinery in biotechnological applications. Commandeering CRISPR adaptation for on-demand memory formation will usher in a new era of biological information storage, with many applications that await discovery.
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Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome

Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14 days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.
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Following nature's roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite multiple numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, we demonstrate the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. We selected a subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high-throughput ELISA platform. Co-expression of the mammalian chaperone BiP, the co-chaperone GRP170, or the peptidyl-prolyl isomerase FKBP2, with the antibody improved specific product yields up to two-fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, we demonstrate that speeding up peptidyl-prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. Our findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast-based antibody factory.
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SSER: Species specific essential reactions database

Essential reactions are vital components of cellular networks. They are the foundations of synthetic biology and are potential candidate targets for antimetabolic drug design. Especially if a single reaction is catalyzed by multiple enzymes, then inhibiting the reaction would be a better option than targeting the enzymes or the corresponding enzyme-encoding gene. The existing databases such as BRENDA, BiGG, KEGG, Bio-models, Biosilico, and many others offer useful and comprehensive information on biochemical reactions. But none of these databases especially focus on essential reactions. Therefore, building a centralized repository for this class of reactions would be of great value.
DESCRIPTION:
Here, we present a species-specific essential reactions database (SSER). The current version comprises essential biochemical and transport reactions of twenty-six organisms which are identified via flux balance analysis (FBA) combined with manual curation on experimentally validated metabolic network models. Quantitative data on the number of essential reactions, number of the essential reactions associated with their respective enzyme-encoding genes and shared essential reactions across organisms are the main contents of the database.
CONCLUSION:
SSER would be a prime source to obtain essential reactions data and related gene and metabolite information and it can significantly facilitate the metabolic network models reconstruction and analysis, and drug target discovery studies. Users can browse, search, compare and download the essential reactions of organisms of their interest through the website http://cefg.uestc.edu.cn/sser .
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EMMA: an Extensible Mammalian Modular Assembly toolkit for the rapid design and production of diverse expression vectors

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility and extensibility of EMMA provide a simple method for production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as: transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries, and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.
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An in vitro synthetic biology platform for the industrial biomanufacturing of myo‐inositol from starch

An in vitro synthetic biology platform for the industrial biomanufacturing of myo‐inositol from starch | SynBioFromLeukipposInstitute | Scoop.it
myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and Inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e. 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale.
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An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch

myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and Inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e. 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale.
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Nucleic acid detection with CRISPR-Cas13a/C2c2

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.
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Translational synthetic biology

Synthetic biology is a recent scientific approach towards engineering biological systems from both pre-existing and novel parts. The aim is to introduce computational aided design approach in biology leading to rapid delivery of useful applications. Though the term reprogramming has been frequently used in the synthetic biology community, currently the technological sophistication only allows for a probabilistic approach instead of a precise engineering approach. Recently, several human health applications have emerged that suggest increased usage of synthetic biology approach in developing novel drugs. This mini review discusses recent translational developments in the field and tries to identify some of the upcoming future developments.
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Mathematization in Synthetic Biology: Analogies, Templates, and Fictions

Mathematization in Synthetic Biology: Analogies, Templates, and Fictions | SynBioFromLeukipposInstitute | Scoop.it
In his famous article “The Unreasonable Effectiveness of Mathematics in the Natural Sciences” Eugen Wigner argues for a unique tie between mathematics and physics, invoking even religious language: “The miracle of the appropriateness of the language of mathematics for the formulation of the laws of physics is a wonderful gift which we neither understand nor deserve” (Wigner 1960: 1). The possible existence of such a unique match between mathematics and physics has been extensively discussed by philosophers and historians of mathematics (Bangu 2012; Colyvan 2001; Humphreys 2004; Pincock 2012; Putman 1975; Steiner 1998). Whatever the merits of this claim are, a further question can be posed with regard to mathematization in science more generally: What happens when we leave the area of theories and laws of physics and move over to the realm of mathematical modeling in interdisciplinary contexts? Namely, in modeling the phenomena specific to biology or economics, for instance, scientists often use methods that have their origin in physics. How is this kind of mathematical modeling justified?
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PeptiGelDesign and regenHU release EMC-Bioink, a new family of 3D bioprinting materials

PeptiGelDesign and regenHU release EMC-Bioink, a new family of 3D bioprinting materials | SynBioFromLeukipposInstitute | Scoop.it
Swiss biomedical company regenHU has partnered with UK-based synthetic biomaterial producer PeptiGelDesign Technologies to release a series of synthetic bioinks for 3D bioprinting. The ready-to-print bioinks were developed for applications in tissue engineering and 3D cell biology.
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Unleashing the Power of Synthetic Proteins

Unleashing the Power of Synthetic Proteins | SynBioFromLeukipposInstitute | Scoop.it
Proteins are the workhorses of all living creatures, fulfilling the instructions of DNA. They occur in a wide variety of complex structures and carry out all the important functions in our body and in all living organisms—digesting food, building tissue, transporting oxygen through the bloodstream, dividing cells, firing neurons, and powering muscles. Remarkably, this versatility comes from different combinations, or sequences, of just 20 amino acid molecules. How these linear sequences fold up into complex structures is just now beginning to be well understood (see box).

Even more remarkably, nature seems to have made use of only a tiny fraction of the potential protein structures available—and there are many. Therein lies an amazing set of opportunities to design novel proteins with unique structures: synthetic proteins that do not occur in nature, but are made from the same set of naturally-occurring amino acids. These synthetic proteins can be “manufactured” by harnessing the genetic machinery of living things, such as in bacteria given appropriate DNA that specify the desired amino acid sequence. The ability to create and explore such synthetic proteins with atomic level accuracy—which we have demonstrated—has the potential to unlock new areas of basic research and to create practical applications in a wide range of fields.

The design process starts by envisioning a novel structure to solve a particular problem or accomplish a specific function, and then works backwards to identify possible amino acid sequences that can fold up to this structure. The Rosetta protein modelling and design software identifies the most likely candidates—those that fold to the lowest energy state for the desired structure. Those sequences then move from the computer to the lab, where the synthetic protein is created and tested—preferably in partnership with other research teams that bring domain expertise for the type of protein being created.

At present no other advanced technology can beat the remarkable precision with which proteins carry out their unique and beautiful functions. The methods of protein design expand the reach of protein technology, because the possibilities to create new synthetic proteins are essentially unlimited. We illustrate that claim with some of the new proteins we have already developed using this design process, and with examples of the fundamental research challenges and areas of practical application that they exemplify:
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A Synthetic Biology Approach to Engineering Living Photovoltaics

A Synthetic Biology Approach to Engineering Living Photovoltaics | SynBioFromLeukipposInstitute | Scoop.it
The ability to electronically interface living cells with electron accepting scaffolds is crucial for the development of next-generation biophotovoltaic technologies. Although recent studies have focused on engineering synthetic interfaces that can maximize electronic communication between the cell and scaffold, the efficiency of such devices is limited by the low conductivity of the cell membrane. This review provides a materials science perspective on applying a complementary, synthetic biology approach to engineering membrane-electrode interfaces. It focuses on the technical challenges behind the introduction of foreign extracellular electron transfer pathways in bacterial host cells and the past and future efforts to engineer photosynthetic organisms with artificial electron-export capabilities for biophotovoltaic applications. The article highlights advances in engineering protein-based, electron-exporting conduits in a model host organism, E. coli, before reviewing state-of-the-art biophotovoltaic technologies that use both unmodified and bioengineered photosynthetic bacteria with improved electron transport capabilities. A thermodynamic analysis is used to propose an energetically feasible pathway for extracellular electron transport in engineered cyanobacteria and identify metabolic bottlenecks amenable to protein engineering techniques. Based on this analysis, an engineered photosynthetic organism expressing a foreign, protein-based electron conduit yields a maximum theoretical solar conversion efficiency of 6-10% without accounting for additional bioengineering optimizations for light-harvesting.
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