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Simulation of E. coli Gene Regulation including Overlapping Cell Cycles, Growth, Division, Time Delays and Noise

Simulation of E. coli Gene Regulation including Overlapping Cell Cycles, Growth, Division, Time Delays and Noise | SynBioFromLeukipposInstitute | Scoop.it
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by
Luo R, Ye L, Tao C, Wang K.


"Due to the complexity of biological systems, simulation of biological networks is necessary but sometimes complicated. The classic stochastic simulation algorithm (SSA) by Gillespie and its modified versions are widely used to simulate the stochastic dynamics of biochemical reaction systems. However, it has remained a challenge to implement accurate and efficient simulation algorithms for general reaction schemes in growing cells. Here, we present a modeling and simulation tool, called 'GeneCircuits', which is specifically developed to simulate gene-regulation in exponentially growing bacterial cells (such as E. coli) with overlapping cell cycles. Our tool integrates three specific features of these cells that are not generally included in SSA tools: 1) the time delay between the regulation and synthesis of proteins that is due to transcription and translation processes; 2) cell cycle-dependent periodic changes of gene dosage; and 3) variations in the propensities of chemical reactions that have time-dependent reaction rates as a consequence of volume expansion and cell division. We give three biologically relevant examples to illustrate the use of our simulation tool in quantitative studies of systems biology and synthetic biology."
http://bit.ly/100FQ9G

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Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging

Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering.
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Programmable Cell Extracts: A New Biomanufacturing Paradigm | University of Cambridge March 16th

Programmable Cell Extracts: A New Biomanufacturing Paradigm | University of Cambridge March 16th | SynBioFromLeukipposInstitute | Scoop.it
Date of Event
16th March 2017
Last Booking Date for this Event
9th March 2017
Places Available
100
Description
6:30pm - 9pm
Thursday 16th March 2017

Bioengineering to produce complex control circuits like diagnostic tests, or to modify metabolic pathways for production of everything from drug and vaccines to flavours and fragrances, has typically taken place in cells that are then grown in large, industrial bioreactors. New methods, using cell extracts that can be programmed quickly and flexibly using DNA, promise a paradigm shift in biomanufacturing and pave the way to novel modes of computational biodesign, rapid prototyping and bioproduction. The opportunity to freeze-dry and ship these biofactories opens up many exciting possibilities for small scale distributed manufacturing, for example just-in-time vaccine production, and has profound implications for emerging bioeconomies.

The Synthetic Biology SRI welcomes two researchers to discuss this new area of synthetic biology and its possible futures.

Dr. Keith Pardee (University of Toronto) works at the interface of synthetic biology and human health. His research focuses on the potential of moving synthetic biology outside of the cell and dry shipment of programmable biofactories to enable diagnostics and just in time production of vaccines and biologics.

Dr. Richard Kelwick (Imperial College) researches cell-free systems and biopolymer production, including establishing cell-free methods and toolkits for new bacterial strains, most recently Bacillus subtilis. He also works on bioreporters and biosensors using synthetic gene circuits.

The talk and dialogue will be followed by a wine reception and delicious finger buffet.

This event is organised by the Synthetic Biology Strategic Research Initiative as part of our Lent Term 2017 SynBio Forum. For more events please visit http://www.synbio.cam.ac.uk/events/forum
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Reactions to CRISPR patent decision | PLOS Synthetic Biology Community

Reactions to CRISPR patent decision | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
This week the US Patent Trial and Appeal Board decided that there is “no interference in fact” between the patent application of Jennifer Doudna and University of California and the patents granted to Feng Zhang and the Broad Institute of Harvard and MIT. You can read the full text of the decision here.

 

Media reaction: Hype!

The immediate reactions put this as a big win for the Broad Institute in as many combative terms as possible. Fight, battle, and war were all used to describe the legal patent interference case being settled and most headlines pronounced the war to be over. Obviously this has been a huge court battle worth enough to merit legal bills going into the tens of millions of dollars, but many experts have noted this is not necessarily the winner-take-all patent fight that many headlines suggest. The interference proceedings were to determine if the set of patents granted to the Broad Institute for CRISPR-Cas9 editing in eukaryotic cells interfere with the patent application from the University of California (UC) that was filed first and tries to generally cover the use of CRISPR-Cas9 in living cells.


Word cloud of 15 top CRISPR patent headlines made using http://wordcloud.cs.arizona.edu/index.html
This decision does mean that barring a successful appeal at the Federal Circuit Appeals Court, the Broad Institute will get to keep the patents using Cas9 to do genome editing in eukaryotic cells–which includes human cells–so that is key to companies that had licensed those patents to develop human therapeutics. The UC patent application tries to make a broader claim to editing in all living cells, which would naturally include eukaryotic and human cells. If UC gets the patent on the broad use in living cells and the Broad has the patents on use in eukaryotic cells, we may still end up in a scenario in which licensing of both patents is necessary to edit eukaryotic cells. Some good reporting that helped me understand this can be found here, here, here, here, and here. After the initial rush to announce the epic end to this patent battle, time allowed for more nuanced and thorough reporting to lay out the remaining uncertainty and caveats.

Market reaction: Follow the hype!

Following the main narrative of the Broad Institute scoring a big win in its ‘epic battle’, the market traders pushed Editas (aligned with Broad) stock about 30% throughout the day and Intellia (aligned with UC) stock dropped nearly 10%. It’s hard for me to tell how much the decision actually has on the value of these companies because it seems like that will depend on how licensing or cross-licensing options work out for any given application. I’ve yet to find much analysis that could definitively map out how the patent licensing will affect medical innovation using CRISPR-Cas9, but that may still be too variable for anyone to know right now.

 

 



There was a lot of discussions of the possible ways this could play out in the biotech and gene editing industries depending on the course that UC takes after the interference defeat. The tweet below from CNBC reporter Meg Tirrell outlines three possible options.
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Stretchable living materials and devices with hydrogel–elastomer hybrids hosting programmed cells

Stretchable living materials and devices with hydrogel–elastomer hybrids hosting programmed cells | SynBioFromLeukipposInstitute | Scoop.it
Living systems, such as bacteria, yeasts, and mammalian cells, can be genetically programmed with synthetic circuits that execute sensing, computing, memory, and response functions. Integrating these functional living components into materials and devices will provide powerful tools for scientific research and enable new technological applications. However, it has been a grand challenge to maintain the viability, functionality, and safety of living components in freestanding materials and devices, which frequently undergo deformations during applications. Here, we report the design of a set of living materials and devices based on stretchable, robust, and biocompatible hydrogel–elastomer hybrids that host various types of genetically engineered bacterial cells. The hydrogel provides sustainable supplies of water and nutrients, and the elastomer is air-permeable, maintaining long-term viability and functionality of the encapsulated cells. Communication between different bacterial strains and with the environment is achieved via diffusion of molecules in the hydrogel. The high stretchability and robustness of the hydrogel–elastomer hybrids prevent leakage of cells from the living materials and devices, even under large deformations. We show functions and applications of stretchable living sensors that are responsive to multiple chemicals in a variety of form factors, including skin patches and gloves-based sensors. We further develop a quantitative model that couples transportation of signaling molecules and cellular response to aid the design of future living materials and devices.
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CRISPR, surrogate licensing, and scientific discovery

Several institutions are embroiled in a legal dispute over the foundational patent rights to CRISPR-Cas9 gene-editing technology, and it may take years for their competing claims to be resolved (1–4). But even before ownership of the patents is finalized, the institutions behind CRISPR have wasted no time capitalizing on the huge market for this groundbreaking technology by entering into a series of license agreements with commercial enterprises (see the figure). With respect to the potentially lucrative market for human therapeutics and treatments, each of the key CRISPR patent holders has granted exclusive rights to a spinoff or “surrogate” company formed by the institution and one of its principal researchers (5, 6). Although this model, in which a university effectively outsources the licensing and commercialization of a valuable patent portfolio to a private company, is not uncommon in the world of university technology transfer, we suggest it could rapidly bottleneck the use of CRISPR technology to discover and develop useful human therapeutics.
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The Birth of CRISPR Inc

Just 5 years ago, the community of researchers studying CRISPR, the powerful new genome editing tool, was small. When the first inklings that CRISPR could become a big business emerged, leading scientists expected to work together. But the attempt at unity collapsed—with a good deal of noise and dust. As the science grew even more compelling and venture capital (VC) beckoned, the jockeying to start CRISPR companies became intense. The research community was rent apart by concerns about intellectual property, academic credit, Nobel Prize dreams, geography, media coverage, egos, personal profit, and loyalty. A billion dollars poured into what might be called CRISPR Inc. from VC firms, pharmaceutical companies, and public stock offerings. And the companies and the academic license holders faced each other down in a battle royale over patents.
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Synthetic biology: Synthetic gene networks that smell

Bioengineers have endowed a consortium of human cells with an artificial sense of smell, enabling the cells to detect, quantify, and remember the presence of gaseous volatile compounds in their environment.
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Constrained Multistate Sequence Design for Nucleic Acid Reaction Pathway Engineering

We describe a framework for designing the sequences of multiple nucleic acid strands intended to hybridize in solution via a prescribed reaction pathway. Sequence design is formulated as a multistate optimization problem using a set of target test tubes to represent reactant, intermediate, and product states of the system, as well as to model crosstalk between components. Each target test tube contains a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Optimization of the equilibrium ensemble properties of the target test tubes implements both a positive design paradigm, explicitly designing for on-pathway elementary steps, and a negative design paradigm, explicitly designing against off-pathway crosstalk. Sequence design is performed subject to diverse user-specified sequence constraints including composition constraints, complementarity constraints, pattern prevention constraints, and biological constraints. Constrained multistate sequence design facilitates nucleic acid reaction pathway engineering for diverse applications in molecular programming and synthetic biology. Design jobs can be run online via the NUPACK web application.
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Synthetic Biology Open Language (SBOL) Version 2.1.0

Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The Synthetic Biology Open Language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.1 of SBOL that builds upon version 2.0 published in last year’s JIB special issue. In particular, SBOL 2.1 includes improved rules for what constitutes a valid SBOL document, new role fields to simplify the expression of sequence features and how components are used in context, and new best practices descriptions to improve the exchange of basic sequence topology information and the description of genetic design provenance, as well as miscellaneous other minor improvements.
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Specifications of Standards in Systems and Synthetic Biology: Status and Developments in 2016

Standards are essential to the advancement of science and technology. In systems and synthetic biology, numerous standards and associated tools have been developed over the last 16 years. This special issue of the Journal of Integrative Bioinformatics aims to support the exchange, distribution and archiving of these standards, as well as to provide centralised and easily citable access to them.
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The Principles of Engineering Immune Cells to Treat Cancer

The Principles of Engineering Immune Cells to Treat Cancer | SynBioFromLeukipposInstitute | Scoop.it
Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart T cell precision therapeutics.
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Synthetic (polymer) biology (membrane): functionalization of polymer scaffolds for membrane proteins

Synthetic (polymer) biology (membrane): functionalization of polymer scaffolds for membrane proteins | SynBioFromLeukipposInstitute | Scoop.it
Highlights

Polymer-based scaffolds functionally present membrane proteins.

Repertoire of polymer-based supports and incorporation methodologies are presented.

Conceptual and technological advances, along with technical challenges are proposed.
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AGOS: A plug-and-play method for the assembly of artificial gene operons into functional biosynthetic gene clusters

The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The plug-and-play method AGOS (Artificial Gene Operon assembly System) was developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a Streptomyces host strain. AGOS was designed as a set of entry plasmids for the construction of artificial gene operons and a SuperCos1 based destination vector, into which the constructed operons can be assembled by Red/ET-mediated recombination. To provide a proof-of-concept of this method, we disassembled the well-known novobiocin biosynthetic gene cluster into four gene operons, encoding for the different moieties of novobiocin. We then genetically reorganized these gene operons with the help of AGOS to finally obtain the complete novobiocin gene cluster again. The production of novobiocin precursors and of novobiocin could successfully be detected by LC-MS and LC-MS/MS. Furthermore we demonstrated that the omission of terminator sequences only had a minor impact on product formation in our system.
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The New Shape of DNA | MIT Spectrum

The New Shape of DNA | MIT Spectrum | SynBioFromLeukipposInstitute | Scoop.it
STEP INTO ASSOCIATE PROFESSOR MARK BATHE’S LAB in the Department of Biological Engineering and you’ll find design and biology merging at the nanoscale. Under an electron microscope you might see a cornucopia of three-dimensional shapes—icosahedra, pyramids, and stars—all assembled from synthetic strands of DNA. “There’s no other molecular medium we can design and fabricate with such a versatility of geometries and precision at the nanoscale as DNA,” says Bathe ’98, SM ’01, PhD ’04.


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DAEDALUS
And Bathe has added to that versatility: with graduate student Sakul Ratanalert, he recently developed software called DAEDALUS, which captures the complex rules of DNA construction in an algorithm that makes three-dimensional DNA design easier and more accessible to a wide range of scientists and engineers.

Most people think of the spiraling set of nucleic acids purely as the code of life. The strings of As, Ts, Cs, and Gs (adenine, thymine, cytosine, and guanine) in cells provide the blueprint for how living things behave and reproduce. And for nearly half a century bioengineers have creatively manipulated those sequences to change the way organisms function—breeding new pest-resistant plants, for example, or microbes that ferment medicines and chemicals.

But the double helix of DNA also possesses unique characteristics as a nano-building material. In 2006, Caltech researcher Paul Rothemund discovered that if he synthesized DNA letters in specific sequences, the molecular bonds that glue the As to Ts and Cs to Gs, and which come undone when DNA replicates, could be used to fold the DNA into two- and three-dimensional shapes. With a nod to both the precision and elegance of the technique, scientists dubbed it “DNA origami.”

The beauty of DNA origami is that once the components are collected, all it takes is a little shake and some Brownian motion—the random movement of particles in fluid—for these shapes to assemble themselves. The system uses a single long strand of DNA as scaffolding on which to stick smaller strings of letters. The DNA conforms to the shape as the letters bond to each other.

The beauty of DNA origami is that once the components are collected, all it takes is a little shake and some Brownian motion—the random movement of particles in fluid—for these shapes to assemble themselves.
But the rules for designing DNA origami are difficult, if not arcane, and lining up nucleotides to fold into corresponding 3-D shapes can tax even the most brilliant minds. “It’s been limited to a small group of experts,” Bathe says. His software is changing that.

Rather than manually fiddling with sequences of nucleotides, DAEDALUS users design the target geometric structures they want, and the algorithm generates the corresponding nucleotide sequences to make them. “You give the software a high-level geometric shape, and then it will automatically produce that shape using DNA,” Bathe says.

In a sense, Bathe himself may be a perfect researcher for exploring how these geometries translate across scales. He’s part of an MIT legacy—the son of longtime engineering faculty member Klaus-Jürgen Bathe. Like his father, the younger Bathe earned his PhD in mechanical engineering; but from childhood, his interests skewed towards biology and medicine. “I’ve always wanted to build technologies that impact human health, more than cars or bridges, like my father,” he says. With DAEDALUS, Bathe has built a bridge of another kind, connecting designers of many disciplines with the tools of molecular biology.

Bathe is now working to harness his DNA nanoshapes to deliver drugs inside the body. Taking a cue from viruses that attach to cells to infect them, Bathe hopes to design a variety of DNA structures that deliver payloads of antibodies or even gene-editing enzymes such as Cas9 to diseased cells within the body. “The holy grail would be to edit the brain for treatment of diseases such as autism or schizophrenia, or cancer cells in malignant tumors,” Bathe says.

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Optogenetic switches for light-controlled gene expression in yeast

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.
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Joint Custody of the Gene Editing Method Crispr Means Someone Is About to Get Rich

Joint Custody of the Gene Editing Method Crispr Means Someone Is About to Get Rich | SynBioFromLeukipposInstitute | Scoop.it
The US Patent Office says two groups, one based at UC Berkeley and the other in Cambridge, Mass. own overlapping parts of a powerful new genetic technique.
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A yellow light for embryo editing

Editing the DNA of a human embryo could be ethically allowable in limited circumstances, says a report released this week by a committee convened by the U.S. National Academy of Sciences and the National Academy of Medicine in Washington, D.C. Such experiments "might be permitted, but only following much more research" on risks and benefits, and "only for compelling reasons and under strict oversight," the group concludes. Those situations could be limited to couples who both have a serious genetic disease and for whom embryo editing is only option for having a healthy biological child. Some researchers are pleased, saying the report is consistent with previous conclusions that altering the DNA of human eggs, sperm, or early embryos—known as germline editing—could be permissible. But others see the report as lowering the bar for embryo editing.
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Evolution of a split RNA polymerase as a versatile biosensor platform

Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.
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Automated Design of Synthetic Cell Classifier Circuits Using a Two-Step Optimization Strategy

Cell classifiers are genetic logic circuits that transduce endogenous molecular inputs into cell-type-specific responses. Designing classifiers that achieve optimal differential response between specific cell types is a hard computational problem because it involves selection of endogenous inputs and optimization of both biochemical parameters and a logic function. To address this problem, we first derive an optimal set of biochemical parameters with the largest expected differential response over a diverse set of logic circuits, and second, we use these parameters in an evolutionary algorithm to select circuit inputs and optimize the logic function. Using this approach, we design experimentally feasible microRNA-based circuits capable of perfect discrimination for several real-world cell-classification tasks. We also find that under realistic cell-to-cell variation, circuit performance is comparable to standard cross-validation performance estimates. Our approach facilitates the generation of candidate circuits for experimental testing in therapeutic settings that require precise cell targeting, such as cancer therapy.
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Synthetic (polymer) biology (membrane): functionalization of polymer scaffolds for membrane proteins

A plethora of polymer-based scaffolds have been designed to facilitate biochemical and biophysical investigation of membrane proteins, with a common goal to stabilize and present them in a functional format. In this review, an up-to-date account of such polymer-based supports and incorporation methodologies are presented. Furthermore, conceptual and imminent technological advances, with associated technical challenges are proposed.
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Implantable Graphene Membranes With Low Cytotoxicity

Implantable Graphene Membranes With Low Cytotoxicity | SynBioFromLeukipposInstitute | Scoop.it
Two-dimensional materials can be formed into enclosures for various substances and a substrate layer can be provided on an outside and/or on an inside of the enclosure, wherein the enclosure is not cytotoxic. The enclosures can be exposed to an environment, such as a biological environment (in vivo or in vitro), where the fibrous layer can promote vascular ingrowth. One or more substances within the enclosure can be released into the environment, one or more selected substances from the environment can enter the enclosure, one or more selected substances from the environment can be prevented from entering the enclosure, one or more selected substances can be retained within the enclosure, or combinations thereof. The enclosure can, for example, allow a sense-response paradigm to be realized. The enclosure can, for example, provide immunoisolation for materials, such as living cells, retained therein.
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Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products

Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products | SynBioFromLeukipposInstitute | Scoop.it
With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the “design-build-test” cycles of biomanufacturing.
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