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Open Biomedical Initiative

Nonprofit Organization for LOW COST, OPEN SOURCE, 3D PRINTABLE Biomedical Technologies
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Patent Law and the Emerging Science of Synthetic Biology: An Examination of Principle and Practice

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Synthetic life and biodiversity

Last year, the first truly novel synthetic life form was created. The Minimal Cell created by the Venter Lab, contains the smallest genome of any known independent organism.[1] While the first synthetic microbe was created in 2010, that was simply a like for like synthetic copy of the genome of an existing bacterium.  Nothing like the Minimal Cell exists in nature.

This great advance in synthetic biology comes at a time where natural life forms are being manipulated in ways never seen before.  The CRISPR gene editing system has been used to create hulk-like dogs, malaria proof mosquitoes, drought resistant wheat and hornless cows. The list of CRISPR-altered animals grows by the month.

Such developments hasten the need for a systematic analysis of the ethics of creating new forms of life. In a recent paper[2], Julian Savulescu and I draw attention to how thoughts regarding the value of biodiversity may bear on this question.

The idea that biodiversity is valuable is ubiquitous. The United Nations “Convention on Biodiversity”, signed by over 160 countries, recognises the “intrinsic value of biological diversity”.[3]  The idea that biodiversity is valuable has also greatly influenced the commercial sector and is a cornerstone of the modern corporate social responsibility movement. The value of biodiversity has even been recognised by the Catholic Church. Pope Francis devotes an entire section of his Encyclical Letter, “On Care For Our Common Home” to the Loss of Biodiversity, describing a new Sin, the destruction of biological diversity.[4]

Most discussions about biodiversity focus on its conservation or protection. Biodiversity is widely seen as a good that should be preserved. We take no stand on whether biodiversity is in fact valuable in this way. Rather we claim that if biodiversity is valuable, this suggests it would be good to increase it, rather than just conserve it at current levels. Just as biodiversity’s value provides reason to prevent species going extinct, it may also provide reasons to introduce novel species; created through synthetic biology or gene editing.

Our claim – that there is no asymmetry between the value of protecting biodiversity, and the value of promoting it (by adding novel species) could be resisted in at least three ways.

One, it could be claimed that our current levels of biodiversity are in some sense optimal. If current levels are optimal then we will have reasons to make sure we do not lose forms of biodiversity, but will not have reasons to create and introduce new life forms.  However, we have strong reasons to doubt the assumption that our current levels of biodiversity are optimal.

Humankind has already had a massive influence on global biodiversity. Recent studies[5] indicate that biodiversity has declined dramatically because of human activity.  Rates of species loss have been accelerated 100 fold in recent centuries. Because we are in a situation where biodiversity has reduced dramatically because of our acts, the claim that current levels are biodiversity are in some sense intrinsically optimal seems very convenient. It would imply that when humans first evolved there was too much biodiversity in the world.

Second, we could appeal to the fragility of nature in attempting to justify conserving, but not promoting biodiversity. A common view is that natural systems, such as ecosystems, are finely balanced and fragile. Because of this, creating new species, but not removing species, is likely to be damaging.

However, such a view of natural systems stems from a misguided view of the causal structure of the natural living world, especially with regard to the interrelations between species that comprise communities and ecosystems.

Whereas it was long-assumed that strategic interaction (e.g. between predator and prey) would lead to evolutionarily stable solutions, there is now a great deal of evidence that biotic interactions will tend to undermine, rather than reinforce, the stability of faunal associations. Hence our current ecosystems are rarely finely balanced, stable communities. Furthermore, research on both living and paleontological communities suggests the impact of a new species moving into an area, tend to be fairly weak in terms of their ecological impact, especially in the context of non-island invasions.

A final way we might resist the claim we have reasons to preserve biodiversity and therefore reason to promote it is an appeal to rights. If species have a right to continued existence, it would be prima facie wrong to contribute to their extinction. However, this wouldn’t imply a duty to create non-existent species.

But it is controversial whether species are the types of entities that could have rights.  While group rights are often proposed for nations, culture groups and so on, they are rarely proposed for species. Only particular groups are considered to have group rights, such as those which show intra-group solidarity, or unity and a sense of shared identity. It is not clear that animal species would meet these conditions. An account of species rights that plausibly defends the view that species have a continue right to existence has not been developed.

In sum, we believe the widely accepted view that biodiversity is a value implies we have reasons to promote biodiversity rather than just conserve it. If biodiversity is in fact valuable, we should be encouraged by recent developments in synthetic biology and genetic engineering which promise to transform life as we know it.

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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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Rational design of an ultrasensitive quorum-sensing switch

One of the purposes of synthetic biology is to develop rational methods that accelerate the design of genetic circuits, saving time and effort spent on experiments and providing reliably predictable circuit performance. We applied a reverse engineering approach to design an ultrasensitive transcriptional quorum-sensing switch. We want to explore how systems biology can guide synthetic biology in the choice of specific DNA sequences and their regulatory relations to achieve a targeted function. The workflow comprises network enumeration that achieves the target function robustly, experimental restriction of the obtained candidate networks, global parameter optimization via mathematical analysis, selection and engineering of parts based on these calculations, and finally, circuit construction based on the principles of standardization and modularization. The performance of realized quorum-sensing switches was in good qualitative agreement with the computational predictions. This study provides practical principles for the rational design of genetic circuits with targeted functions.
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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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