Realizing the value of synthetic biology in biotechnology and medicine requires the design of molecules with specialized functions. Due to its close structure to function relationship, and the availability of good structure prediction methods and energy models, RNA is perfectly suited to be synthetically engineered with predefined properties. However, currently available RNA design tools cannot be easily adapted to accommodate new design specifications. Furthermore, complicated sampling and optimization methods are often developed to suit a specific RNA design goal, adding to their inflexibility. RESULTS: We developed a C ++ library implementing a graph coloring approach to stochastically sample sequences compatible with structural and sequence constraints from the typically very large solution space. The approach allows to specify and explore the solution space in a well defined way. Our library also guarantees uniform sampling, which makes optimization runs performant by not only avoiding reevaluation of already found solutions, but also by raising the probability of finding better solutions for long optimization runs. We show that our software can be combined with any other software package to allow diverse RNA design applications. Scripting interfaces allow the easy adaption of existing code to accommodate new scenarios, making the whole design process very flexible. We implemented example design approaches written in Python to demonstrate these advantages. AVAILABILITY: RNAblueprint, Python implementations and benchmark data sets are available at github: https://github.com/ribonets /.
With the increasingly dominant role of smartphones in our lives, mobile health care systems integrating advanced point-of-care technologies to manage chronic diseases are gaining attention. Using a multidisciplinary design principle coupling electrical engineering, software development, and synthetic biology, we have engineered a technological infrastructure enabling the smartphone-assisted semiautomatic treatment of diabetes in mice. A custom-designed home server SmartController was programmed to process wireless signals, enabling a smartphone to regulate hormone production by optically engineered cells implanted in diabetic mice via a far-red light (FRL)–responsive optogenetic interface. To develop this wireless controller network, we designed and implanted hydrogel capsules carrying both engineered cells and wirelessly powered FRL LEDs (light-emitting diodes). In vivo production of a short variant of human glucagon-like peptide 1 (shGLP-1) or mouse insulin by the engineered cells in the hydrogel could be remotely controlled by smartphone programs or a custom-engineered Bluetooth-active glucometer in a semiautomatic, glucose-dependent manner. By combining electronic device–generated digital signals with optogenetically engineered cells, this study provides a step toward translating cell-based therapies into the clinic.
As the Synthetic Biology Open Language (SBOL) data and visual standards gain acceptance for describing genetic designs in a detailed and reproducible way, there is an increasing need for an intuitive sequence editor tool that biologists can use that supports these standards. This paper describes SBOLDesigner 2, a genetic design automation (GDA) tool that natively supports both the SBOL data model (Version 2.1) and SBOL Visual (Version 1.0). This software is enabled to fetch and store parts and designs from SBOL repositories, such as SynBioHub. It can also import and export data about parts and designs in FASTA, GenBank, and SBOL 1.1 data format. Finally, it possesses a simple and intuitive user interface. This paper describes the design process using SBOLDesigner 2, highlighting new features over the earlier prototype versions. SBOLDesigner 2 is released freely and open source under the Apache 2.0 license.
Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action-the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.
The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a "brute force" validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. RESULTS: We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 "dispensable" genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. CONCLUSIONS: This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.
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.
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.
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.
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.
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.
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.
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.
The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis.
The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.
New strategies to control cholera are urgently needed. This study develops an in vitro proof-of-concept sense-and-eradicate system in a wild-type Escherichia coli strain to target the causative pathogen Vibrio cholerae using a synthetic biology approach. Our engineered E. coli specifically detects V. cholerae via its quorum-sensing molecule CAI-1 and responds by expressing the lysis protein (YebF-Art-085), thereby self-lysing to release the killing protein (Art-085) to eradicate V. cholerae. For this report, we individually characterized the YebF-Art-085 and Art-085 expression and their activities when coupled to our previously developed V. cholerae biosensing circuit. We show that in the presence of V. cholerae supernatant, the final integrated sense-and-eradicate system in our engineered E. coli can inhibit the growth of V. cholerae cells effectively. This work represents the first step toward a novel probiotic treatment modality that could potentially prevent and treat cholera in the future.
Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength- and intensity-dependent photoconversion, signaling, and output gene expression for our two previously engineered light-sensing Escherichia coli two-component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open-source "Light Plate Apparatus" device. In principle, the parameterized model should predict the gene expression response to any time-varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross-reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision-making pathways integrate multiple input signals.
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. 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, 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”. 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.
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 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.
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.
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.
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 .
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.
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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