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Application of Synthetic Biology for Increasing Anaerobic Biodiesel Production in Escherichia coli

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Gerd Moe-Behrens's insight:

Master Thesis

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Michael Christopher Wierzbicki 

 

http://bit.ly/HSz8g0

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Synthetic Biology Market is expected to Grow more than US$ 38 Billion by 2020 

Synthetic Biology Market is expected to Grow more than US$ 38 Billion by 2020  | SynBioFromLeukipposInstitute | Scoop.it
New York, September 29: Market Research Engine has published a new report titled as “Synthetic Biology Market (Synthetic DNA, Synthetic Genes, Synthetic Cells, XNA, Chassis Organisms, DNA Synthesis, Oligonucleotide Synthesis) - Global Industry...
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Twister ribozymes as highly versatile expression platforms for artificial riboswitches

Twister ribozymes as highly versatile expression platforms for artificial riboswitches | SynBioFromLeukipposInstitute | Scoop.it
The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.
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Synthetic Biology Publications: Top Ten

Synthetic Biology Publications: Top Ten | SynBioFromLeukipposInstitute | Scoop.it
The latest insights and analyses from SynbiCITE – synthetic biology industrial accelerator based at Imperial College, London, UK
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Synthetic Biology-Based Point-of-Care Diagnostics for Infectious Disease

Synthetic Biology-Based Point-of-Care Diagnostics for Infectious Disease | SynBioFromLeukipposInstitute | Scoop.it
Infectious diseases outpace all other causes of death in low-income countries, posing global health risks, laying stress on healthcare systems and societies, and taking an avoidable human toll. One solution to this crisis is early diagnosis of infectious disease, which represents a powerful way to optimize treatment, increase patient survival rate, and decrease healthcare costs. However, conventional early diagnosis methods take a long time to generate results, lack accuracy, and are known to seriously underperform with regard to fungal and viral infections. Synthetic biology offers a fast and highly accurate alternative to conventional infectious disease diagnosis. In this review, we outline obstacles to infectious disease diagnostics and discuss two emerging alternatives: synthetic viral diagnostic systems and biosensors. We argue that these synthetic biology-based approaches may overcome diagnostic obstacles in infectious disease and improve health outcomes.
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Human-Computer Interaction 

Human-Computer Interaction  | SynBioFromLeukipposInstitute | Scoop.it
Human-Computer Interaction - HCI Research. 21,477 likes · 273,246 talking about this. A free page for sharing and collecting latest informatio
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Targeted Gene Activation Using RNA-Guided Nucleases

The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.
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A global genetic interaction network maps a wiring diagram of cellular function

A global genetic interaction network maps a wiring diagram of cellular function | SynBioFromLeukipposInstitute | Scoop.it
INTRODUCTION
Genetic interactions occur when mutations in two or more genes combine to generate an unexpected phenotype. An extreme negative or synthetic lethal genetic interaction occurs when two mutations, neither lethal individually, combine to cause cell death. Conversely, positive genetic interactions occur when two mutations produce a phenotype that is less severe than expected. Genetic interactions identify functional relationships between genes and can be harnessed for biological discovery and therapeutic target identification. They may also explain a considerable component of the undiscovered genetics associated with human diseases. Here, we describe construction and analysis of a comprehensive genetic interaction network for a eukaryotic cell.
RATIONALE
Genome sequencing projects are providing an unprecedented view of genetic variation. However, our ability to interpret genetic information to predict inherited phenotypes remains limited, in large part due to the extensive buffering of genomes, making most individual eukaryotic genes dispensable for life. To explore the extent to which genetic interactions reveal cellular function and contribute to complex phenotypes, and to discover the general principles of genetic networks, we used automated yeast genetics to construct a global genetic interaction network.
RESULTS
We tested most of the ~6000 genes in the yeast Saccharomyces cerevisiae for all possible pairwise genetic interactions, identifying nearly 1 million interactions, including ~550,000 negative and ~350,000 positive interactions, spanning ~90% of all yeast genes. Essential genes were network hubs, displaying five times as many interactions as nonessential genes. The set of genetic interactions or the genetic interaction profile for a gene provides a quantitative measure of function, and a global network based on genetic interaction profile similarity revealed a hierarchy of modules reflecting the functional architecture of a cell. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections associated with defects in cell cycle progression or cellular proteostasis. Importantly, the global network illustrates how coherent sets of negative or positive genetic interactions connect protein complex and pathways to map a functional wiring diagram of the cell.
CONCLUSION
A global genetic interaction network highlights the functional organization of a cell and provides a resource for predicting gene and pathway function. This network emphasizes the prevalence of genetic interactions and their potential to compound phenotypes associated with single mutations. Negative genetic interactions tend to connect functionally related genes and thus may be predicted using alternative functional information. Although less functionally informative, positive interactions may provide insights into general mechanisms of genetic suppression or resiliency. We anticipate that the ordered topology of the global genetic network, in which genetic interactions connect coherently within and between protein complexes and pathways, may be exploited to decipher genotype-to-phenotype relationships.

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Synthetic Biology-Based Point-of-Care Diagnostics for Infectious Disease

Infectious diseases outpace all other causes of death in low-income countries, posing global health risks, laying stress on healthcare systems and societies, and taking an avoidable human toll. One solution to this crisis is early diagnosis of infectious disease, which represents a powerful way to optimize treatment, increase patient survival rate, and decrease healthcare costs. However, conventional early diagnosis methods take a long time to generate results, lack accuracy, and are known to seriously underperform with regard to fungal and viral infections. Synthetic biology offers a fast and highly accurate alternative to conventional infectious disease diagnosis. In this review, we outline obstacles to infectious disease diagnostics and discuss two emerging alternatives: synthetic viral diagnostic systems and biosensors. We argue that these synthetic biology-based approaches may overcome diagnostic obstacles in infectious disease and improve health outcomes.
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In a first, 12 Pakistani students set to compete in iGEM world championship 

In a first, 12 Pakistani students set to compete in iGEM world championship  | SynBioFromLeukipposInstitute | Scoop.it
The studen­ts will use synthe­tic biolog­y to solve one of the most pressi­ng enviro­nmenta­l challe­nges facing the countr­y
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Uppsala University iGEM - Timeline 

Uppsala University iGEM, Uppsala (Uppsala, Sweden). 875 likes · 20 talking about this. Synthetic biology is the new revolutionizing field o
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Synthetic biology competition launched

Synthetic biology competition launched | SynBioFromLeukipposInstitute | Scoop.it
An annual competition has been launched to assist companies aiming to solve world issues with synthetic biology.
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Just add water: Biomolecular manufacturing ‘on-the-go’

Wyss Institute team unveils a low-cost, portable method to manufacture biomolecules for a wide range of vaccines, other therapies as well as diagnostics

(BOSTON) — Even amidst all the celebrated advances of modern medicine, basic life-saving interventions are still not reaching massive numbers of people who live in our planet’s most remote and non-industrialized locations. The World Health Organization states that one half of the global population lives in rural areas. And according to UNICEF, last year nearly 20 million infants globally did not receive what we would consider to be basic vaccinations required for a child’s health.

These daunting statistics are largely due to the logistical challenge of transporting vaccines and other biomolecules used in diagnostics and therapy, which conventionally require a "cold chain" of refrigeration from the time of synthesis to the time of administration. In remote areas lacking power or established transport routes, modern medicine often cannot reach those who may need it urgently.
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Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology

Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology | SynBioFromLeukipposInstitute | Scoop.it
Engineering the Microbiome: Using Synthetic Biology as the Interface Between Ourselves and our Ecology
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Unintended Consequences of Expanding the Genetic Alphabet

Unintended Consequences of Expanding the Genetic Alphabet | SynBioFromLeukipposInstitute | Scoop.it
The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world’s first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymatic activities. Importantly, however, there is currently no knowledge of the photochemical properties of d5SICS or dNaM—properties that are central to the chemical integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside’s excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.
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New research center is dedicated to engineering cells into living machines

New research center is dedicated to engineering cells into living machines | SynBioFromLeukipposInstitute | Scoop.it
The Golden State will soon house its own “blue-sky” bioengineering center thanks to a healthy grant from the National Science Foundation.
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Materials Ecology: design that works with and is inspired by nature.

Materials Ecology: design that works with and is inspired by nature. | SynBioFromLeukipposInstitute | Scoop.it

Really cool concept of "materials ecology" in design/architecture 

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The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field 

The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field  | SynBioFromLeukipposInstitute | Scoop.it
Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This viewpoint article underscores the need for new bacterial chassis, as well as discusses some of the important considerations in their selection. It also highlights a few examples of robust, tractable bacterial species that can meet the demands of tomorrow’s state-of-the-art in SB. Significant advances have been made in the first 15 years since this field has emerged. However, the advances over the next 15 years will occur not in laboratory organisms, but in fieldable species where the potential of SB can be fully realized in game changing technology.
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Alternative Watson-Crick Synthetic Genetic Systems

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
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Inspired by Nature 

Inspired by Nature  | SynBioFromLeukipposInstitute | Scoop.it
Retooling Biological Systems To Develop New Drugs And Therapies
Despite all that humankind has created, the natural engineering of biological systems never ceases to amaze. The human brain continues to outmatch manmade machines when it comes to pattern recognition: Finding the elusive “Where’s Waldo” is a breeze for us but not so much for artificial intelligence. And plants found in nature – simple organisms capable of making complex molecules – remain the basis for many drugs used today.
While drawing inspiration from “living” technology is far from new, building novel biological systems is here and now. An emerging discipline, synthetic biology brings together concepts from engineering, physics and computer science to create artificial biological processes to improve on nature’s original design. Much of the work in this fledgling field has centered on reprogramming cells by modifying their genetic code (or DNA) to serve specific purposes. Scientists are exploring innovative “synbio” processes that offer less expensive and faster methods for developing novel products, from environmentally-friendly fuel to reengineered immune cells that fight cancer.
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New Map Finally Reveals A Global Genetic Network Of Life

New Map Finally Reveals A Global Genetic Network Of Life | SynBioFromLeukipposInstitute | Scoop.it
Researchers at the University of Toronto’s Donnelly Centre have created the first map that shows the global genetic interaction network of a cell. It begins to explain how thousands of genes coordinate with one another to orchestrate cellular life.

The study was led by U of T Professors Brenda Andrews and Charles Boone, and Professor Chad Myers of the University of Minnesota-Twin Cities. It opens the door to a new way of exploring how genes contribute to disease with a potential for developing finely-tuned therapies. The findings are published in the journal Science.



“We’ve created a reference guide for how to chart genetic interactions in a cell,” said Michael Costanzo, a research associate in the Boone lab and one of the researchers who spearheaded the study. “We can now tell what kind of properties to look for in searching for highly connected genes in human genetic networks with the potential to impact genetic diseases.”


The study took 15 years to complete and adds to Andrews’ rich scientific legacy for which she was awarded a Companion of the Order of Canada.

Just as societies in the world are organized from countries down to local communities, the genes in cells operate in hierarchical networks to organize cellular life. Researchers believe that if we are to understand what 20,000 human genes do, we must first find out how they are connected to each other.

Studies in yeast cells first showed the need to look farther than a gene’s individual effect to understand its role. With 6,000 genes, many of which are also found in humans, yeast cells are a relatively simple but powerful stand-ins for human cells.

Over a decade ago, an international consortium of scientists first deleted every yeast gene, one by one. They were surprised to find that only one in five were essential for survival. It wasn’t until last year that advances in gene-editing technology allowed scientists to tackle the equivalent question in human cells. It revealed the same answer: a mere fraction of genes are essential in human cells too.

These findings suggested most genes are “buffered” to protect the cell from mutations and environmental stresses. To understand how this buffering works, scientists had to ask if cells can survive upon losing more than one gene at a time, and they had to test millions of gene pairs.

Andrews, Boone and Myers led the pioneering work in yeast cells by deleting two genes at a time in pair combinations. They were trying to look for gene pairs that are essential for survival. This called for custom-built robots and a state-of-the-art automated pipeline to analyse almost all of the mind-blowing 18 million different combinations.

The yeast map identified genes that work together in a cell. It shows how, if a gene function is lost, there’s another gene in the genome to fill its role. Consider a bicycle analogy: a wheel is akin to an essential gene – without it, you couldn’t ride the bike. But front brakes? Well, as long as the back brakes are working, you might be able to get by. But if you were to lose both sets of brakes, you are heading for trouble.

Geneticists say that front and back brakes are “synthetic lethal,” meaning that losing both – but not one – spells doom. Synthetic lethal gene pairs are relatively rare, but because they tend to control the same process in the cell, they reveal important information about genes we don’t know much about. For example, scientists can predict what an unexplored gene does in the cell simply based on its genetic interaction patterns.

It’s becoming increasingly clear that human genes also have one or more functional backups. So researchers believe that instead of searching for single genes underlying diseases, we should be looking for gene pairs. That is a huge challenge because it means examining about 200 million possible gene pairs in the human genome for association with a disease.

Fortunately, with the know-how from the yeast map, researchers can now begin to map genetic interactions in human cells and even expand it to different cell types. Together with whole-genome sequences and health parameters measured by new personal devices, it should finally become possible to find combinations of genes that underlie human physiology and disease.



“Without our many years of genetic network analysis with yeast, you wouldn’t have known the extent to which genetic interactions drive cellular life or how to begin mapping a global genetic network in human cells,” said Boone, who is also a professor in U of T’s molecular genetics department and a co-director of the Genetic Networks program at the Canadian Institute for Advanced Research (CIFAR) and holds Canada Research Chair in Proteomics, Bioinformatics and Functional Genomics. We have tested the method to completion in a model system to provide the proof of principle for how to approach this problem in human cells. There’s no doubt it will work and generate a wealth of new information."


The concept of synthetic lethality is already changing cancer treatment because of its potential to identify drug targets that exist only in tumour cells. Cancer cells differ from normal cells in that they have scrambled genomes littered with mutations. They’re like a bicycle without a set of brakes. If scientists could find the highly vulnerable back-up genes in cancer, they could target specific drugs at them to destroy only the cells that are sick, leaving the healthy ones untouched.
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Portable, On-Demand Biomolecular Manufacturing

Portable, On-Demand Biomolecular Manufacturing | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology applies rational design principles of engineering to molecular biology to build genetic devices, which have begun to impact the diagnostic and therapeutic space. This approach has helped to create whole-cell biosensors (Kobayashi et al., 2004), genetically modified probiotics (Danino et al., 2015), and a growing capability for cell-based biomolecular manufacturing. Rooted in genetically engineered production cell lines, biosynthesis is increasingly a mainstay for industrial drug production (Fossati et al., 2014), protein therapeutics (Dimitrov, 2012), fuels (Torella et al., 2015), and other commodities (Chubukov et al., 2016). However, the reliance on living cellular hosts to operate the genetic programs that underpin biosynthesis is accompanied by biosafety regulations, practical hurdles, and specialized skills that limit their operation to laboratory settings. Therefore, vaccines and other protein-based biomolecules must be globally distributed from centralized foundries and, most often, require a cold chain for stability. These limitations impact distribution costs and highlight the challenge of delivering the benefits of these technologies to developing regions. We recently reported a method for the safe deployment of genetically encoded tools (Pardee et al., 2014). Using freeze-dried, cell-free (FD-CF) expression machinery on paper, we generated a platform that retains the fundamental protein synthesis capability of live cells while remaining abiotic, sterile, and portable. In combination with toehold switch RNA sensors (Green et al., 2014), this platform was used to demonstrate a new class of low-cost diagnostic tools (Pardee et al., 2016).

FD-CF reactions offer additional venues for the distributed use of synthetic biology apart from diagnostics, such as the exciting prospect of the portable manufacture of pharmaceuticals, therapeutic proteins, and other biomolecules. In recent years, in vitro biosynthesis from fresh or frozen lysates has developed remarkably, including the biomanufacture of difficult molecules that cause cell toxicity and the incorporation of non-canonical elements (Amiram et al., 2015, Dudley et al., 2015, Karim and Jewett, 2016, Sullivan et al., 2016, Welsh et al., 2012, Zawada et al., 2011). These advances have thus far been tied to laboratory settings where the necessary skills and equipment are found. Building off of this foundation, the proposed use of FD-CF systems, with their long-term activity at room temperature (>1 year) and ease of operation, could alleviate both the restrictions of live-cell biosynthesis and cold-chain distribution requirements (Pardee et al., 2014). Recent reports draw emphasis to a pressing need for the decentralization of therapeutic biomanufacturing, offering novel alternatives that, nonetheless, require expensive, large equipment and highly skilled operators or yet rely on production from living cells (Adamo et al., 2016, Perez-Pinera et al., 2016). Previous work has demonstrated protein production from lyophilized reactions, which strongly supports the notion of advancing this concept further toward on-demand, local biomanufacturing (Salehi et al., 2016, Smith et al., 2014). In addition to portability, the FD-CF format has all of the advantages that are innate to in vitro biosynthesis. Moreover, with buffers, cellular machinery, and molecular instructions all compressed into a single FD reaction pellet, on-demand, on-site activation would only require the addition of water and yields product within 1–2 hr, without the need for specialized equipment and skill. This system could be applied for global health and personalized medicine, making scalable molecular synthesis available to anyone with FD reagents and DNA-encoding biosynthesis instructions.

Here, we present a series of vignettes describing the production of a diverse set of therapeutics and molecular tools for clinical and research environments using FD-CF reaction pellets (Figure 1). Nested within this proof of concept is a drive to create inexpensive alternatives for developing world applications where cost is a major factor to access. We begin with the production of two therapeutic classes of molecules: antimicrobial peptides (AMPs) and vaccines. For the former, we demonstrate purification schemes and validate antimicrobial activity. For the latter, we verify the expression of three vaccine antigens, including the scaled-up production and functional characterization of the diphtheria toxoid antigen (DT), which is administered to an animal model to confirm a successful immune response. Next, we establish a novel combinatorial approach to generate 90 possible affinity conjugates for applications in research and healthcare, of which a subset are functionally validated. Finally, we reconstitute a multi-enzyme biosynthesis pathway for small-molecular therapeutic production and offer a mix-and-match method to synthesize multiple pathway products, which are confirmed using mass spectrometry (MS).
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Portable, On-Demand Biomolecular Manufacturing

Portable, On-Demand Biomolecular Manufacturing | SynBioFromLeukipposInstitute | Scoop.it
Ready-to-use preparations enable on-site, on-demand production of biomolecules like
antimicrobials and vaccines without refrigeration or specialized equipment.
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MIT-Led Team Creates Freeze-Dried Cellular Components to Produce Biopharmaceuticals on Demand

MIT-Led Team Creates Freeze-Dried Cellular Components to Produce Biopharmaceuticals on Demand | SynBioFromLeukipposInstitute | Scoop.it
A team of researchers at MIT and other institutions have developed miniature freeze-dried pellets that possess all of the molecular machinery required to convert DNA into proteins, which could form the basis for on-demand production of vaccines and...
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Synthetic Biology: Innovative approaches for pharmaceutics and drug delivery

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