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On "Join Papester Collective 1.0: How to reply to #icanhazpdf in 3 seconds"

On "Join Papester Collective 1.0: How to reply to #icanhazpdf in 3 seconds" | SynBioFromLeukipposInstitute | Scoop.it
Gerd Moe-Behrens's insight:

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+Eugenio Battaglia 

"I'm totally supporting this potential system theorized some days ago by Micah Allen and his friend Hauke on Allen's Neuroconscience blog . They discuss a quick and reliable strategy to share papers behind a paywall.

The proposed system is really easy and accessible by everyone, since it uses particular twitter's #hashtags for query and response.I strongly believe that what started after Aaron Swartz's dead with #pdftribute, and continued with #sharecredentials (unfortunately and strangely still not so shared on twitter), and now followed by #icanhazpdf / #papester  will quickly lead to a massive weaken of paywall systems. Therefore, this will push people to understand and to propose alternative ways that are more ethically correct and also apt to current science needs.  Also, i don't believe that this ways are so illegal to warn people not to use them, and even if they are is just because old rules simply doesn't fit anymore with the actual need of a more open and accessible science. Finally, i could suggest you to leave the army and join the pirates! Because no one will be interested in fighting for your right of research. We are facing really big issues nowadays, and without a coordinated network strategy we won't be able to solve this topics together. Instead we would leave this solutions to big institutions and corporations, that to this date are too biased by market logics and profitable goals.Concluding, i think this battle has to be fight by those who believe that knowledge has to be definitely accessible to everyone on Earth. I don't want to necessarily appear as a criminal, and of course I'm aware that editors have to make profits in order to produce a quality service, but the results of paywalls are pretty clear. A recent article on Cyberpsychology, behavior and social networking journal, argue:"In a national online longitudinal survey, participants reported their attitudes and behaviors in response to the recently implemented metered paywall by the New York Times. Previously free online content now requires a digital subscription to access beyond a small free monthly allotment. Participants were surveyed shortly after the paywall was announced and again 11 weeks after it was implemented to understand how they would react and adapt to this change. Most readers planned not to pay and ultimately did not. Instead, they devalued the newspaper, visited its Web site less frequently, and used loopholes, particularly those who thought the paywall would lead to inequality."[1]Finally, I want share the interesting idea of Allen and his friend Hauke:"Yesterday my friend Hauke and I theorized about a kind of dream scenario- a totally distributed, easy to use, publication liberation system. This is perhaps not feasible at this point [1]. Today we’re going to present something that will be useful right now. The essential goal here is to make it so that anyone, anywhere, can access the papers they need in a timely manner. The idea is to take advantage of existing strategies and tools to streamline paper sharing as much as possible. Folks already do this- every day on twitter or in private, requests for papers are made and fulfilled. Our goal is to completely streamline this process down to a few clicks of your mouse. That way a small but dedicated group of folks – the Papester Collective – can ensure that #icanhazpdf requests are fulfilled almost instantly. This is a work in progress. Leave comments on how to improve and further streamline this system and join the collective!  SHORT VERSION: HOW TO GET A PAPER BEHIND A PAYWALL QUICKLY Tweet (for example): “#icanhazpdf http://dx.doi.org/10.1523/JNEUROSCI.4568-12.2013” Show your support for the papester collective by tweeting: “Thanks to #papester, my #icanhazpdf request was fufilled in seconds! Join: http://bit.ly/W7fa2T #pdftribute”Click: Here you can find more detailed instructions. HOW TO JOIN THE COLLECTIVE AND START SERVING REQUESTSSHORT INSTRUCTIONS AND REQUIRED SOFTWARE:Twitter:  Monitor #icanhazpdf #requests Zotero and zotero browser plugin: after clicking on DOI link or abstract page just click on ‘Save to Zotero’ button to auto-grabs PDFs  Zotfile: automatically copies new Zotero pdfs files saved to public Dropbox folder  Dropbox: Cloud storage system to seamlessly share files with anyone without login. Dropbox linker: automatically adds links from public folder to your clipboard Reply to request tweets: paste URL from clipboard and if you want #papester That’s it! Now you can just click request links, click the Zotero get PDF button, and CTRL+V a dropbox direct download link in response! Click: Here you can find more detailed instructions.  1.The fundamental problem: uploading huge repositories of scientific papers is not sensible for now. It’s too much data (50 million papers * 0.5-1.5 megabytes together make up ~ 25-75 Terrabytes) and the likelihood for every paper to be downloaded is more uniformly distributed than with files traditionally shared like music. For instance, there are 100 million songs x 3.5 mb songs, and it is difficult to find exotic songs online – some songs have decent availability now because there are only a few favourites – not so with favourite papers. Also, fewer people will share papers than songs, so this makes it more even more difficult to sustain a complete repository. Thus, we need a system that fufills requests individually. Disclaimer: Please make sure you only share papers with friends who also have the copyrights to the papers you share." From Michah Allen's Neuroscience BlogAs you can see I reported the disclaimer of Allen's Blog. I don't agree with that, and also i don't see how my #sharecredentials is far more illegal in this sense! Free your paper, Free your credentials and share your knowledge for free! use the #sharecredentials to share your credentials for limited-access data-bases use the #pdftribute to share your papers by using the bookmarklet here http://aaronsw.archiveteam.org/ and your JSTOR account you can "liberate" one paperTweet #icanhazpdf  ARTICLE_URL or DOI;  more info hereCook, J., & Attari, S. (2012). Paying for What Was Free: Lessons from the PaywallCyberpsychology, Behavior, and Social Networking, 15 (12), 682-687 DOI: 10.1089/cyber.2012.0251


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CRISPR/Cas9 Reveals Cancer’s Synthetic Lethal Vulnerabilities

CRISPR/Cas9 Reveals Cancer’s Synthetic Lethal Vulnerabilities | SynBioFromLeukipposInstitute | Scoop.it
The CRISPR/Cas9 gene-editing system has been used to identify more than 120 synthetic-lethal gene interactions in cancer cells. These interactions could guide drug developers to new combination therapies that could selectively kill cancer cells and spare healthy cells.

Synthetic-lethal gene interactions may occur when certain pairs of mutated genes are present. When there is a mutation in either of these genes within a cell, the cell remains viable. But when there are mutations in both genes, the result is cell death. Synthetic-lethal gene interactions are especially important in the context of cancer therapies. If at least one of the genes in the interaction is specific to cancer, then a drug that inhibits the other gene would selectively kill only cancer cells.

The synthetic-lethal concept has been around for years, but it has been underdeveloped because chemical and genetic tools for the perturbation of gene function in somatic cells have been lacking. But this limitation has been addressed by researchers at the University of California, San Diego. These researchers report that they developed a new method to search for synthetic-lethal gene combinations.

The method appeared March 20 in the journal Nature Methods, in an article entitled “Combinatorial CRISPR–Cas9 Screens for De Novo Mapping of Genetic Interactions.”

“We developed a systematic approach to map human genetic networks by combinatorial CRISPR–Cas9 perturbations coupled to robust analysis of growth kinetics,” wrote the article’s authors. “We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction.”

In this article, the UC San Diego team described how they used the gene-editing technique CRISPR/Cas9 to simultaneously test for thousands of synthetic-lethal interactions. The researchers designed a CRISPR/Cas9 system with two guide RNAs: (1) one that targets a tumor suppressor gene that is commonly mutated in cancer and (2) one that targets a gene that could also be disrupted by a cancer drug. They deployed this system against 73 genes in three laboratory cell lines—human cervical cancer, lung cancer, and embryonic kidney cells. Then they measured cell growth and death.

“Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision,” the authors noted. “From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.”

"The ovarian cancer drug olaparib works by synthetic lethality—it inhibits a gene that, when a BRCA gene is also mutated, kills just those cancer cells," said John Paul Shen, M.D., clinical instructor and postdoctoral fellow at UC San Diego School of Medicine and Moores UCSD Cancer Center. "Many other cancers could likely be treated this way as well, but we don't yet know which gene mutation combinations will be synthetic-lethal."

"Identifying underlying genetic interactions in this way can reveal important functional relationships between genes, such as contributions to the same protein complex or pathway," co-senior author Trey Ideker, Ph.D., professor in the UC San Diego School of Medicine, founder of the UC San Diego Center for Computational Biology and Bioinformatics and co-director of the Cancer Cell Map Initiative. "This in turn can impact both our fundamental understanding of biological systems, as well as therapeutics development."

Many of the gene interactions the team identified were synthetic-lethal in just one of the three cell lines tested. This means that synthetic-lethal interactions may be different in different types of cancer. The researchers said this will be an important consideration for future drug development.

"Moving forward, we intend to further refine our technology platform and make it more robust," said co-senior author Prashant Mali, Ph.D., assistant professor in the Jacobs School of Engineering at UC San Diego. "And we are scaling our cancer genetic networks maps so we can systematically identify new combination therapies."
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EmptPhage engineering: how advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophagesy title

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Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942

Cyanobacteria are oxygenic photosynthetic prokaryotes that are able to assimilate CO2 using solar energy and water. Metabolic engineering of cyanobacteria has suggested the possibility of direct CO2 conversion to value-added chemicals. However, engineering of cyanobacteria has been limited due to the lack of various genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for biochemicals from CO2. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942 as a model cyanobacterium. The SyneBrick chromosomal integration vectors provide three inducible expression systems to control gene expression and three neutral sites for chromosomal integrations. Using a SyneBrick vector, LacI-regulated gene expression led to 24-fold induction of the eYFP reporter gene with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer in S. elongatus PCC 7942 under 5% (v/v) CO2. TetR-regulated gene expression led to 19-fold induction of the GFP gene when 100 nM anhydrotetracycline (aTc) inducer was used. Gene expression decreased after 48 h due to degradation of aTc under light. T7 RNA polymerase-based gene expression resulted in efficient expression with a lower IPTG concentration than a previously developed pTrc promoter. A library of T7 promoters can be used for tunable gene expression. In summary, SyneBrick vectors were developed as a synthetic biology platform for gene expression in S. elongatus PCC 7942. These results will accelerate metabolic engineering of biosolar cell factories through expressing and controlling multiple genes of interest.
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Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.
RESULTS:
In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.
CONCLUSION:
Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.
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DNA Strand-Displacement Timer Circuits 

RT @ACSSynBio: DNA Strand-Displacement Timer Circuits

https://t.co/xHjNdwJDyj https://t.co/Nkb5DQouch
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Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics

Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics.
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Engineering and Characterizing Synthetic Protease Sensors and Switches

Proteases are finding an increasing number of applications as molecular tools and reporters in biotechnology and basic research. Proteases are also increasingly incorporated into synthetic genetic signaling circuits equipping cells with tailored new functions. In the majority of cases however, proteases are employed in constitutively active forms which limits their utility and application as molecular sensors. The following chapter provides a detailed experimental protocol for converting constitutively active proteases into regulated protease receptors. Such receptors can potentially sense, transduce, and amplify any molecular input, thereby opening up a range of new applications in basic research, biotechnology, and synthetic biology.
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Synthetic Protein Switches: Theoretical and Experimental Considerations

Synthetic protein switches with tailored response functions are finding increasing applications as tools in basic research and biotechnology. With a number of successful design strategies emerging, the construction of synthetic protein switches still frequently necessitates an integrated approach that combines detailed biochemical and biophysical characterization in combination with high-throughput screening to construct tailored synthetic protein switches. This is increasingly complemented by computational strategies that aim to reduce the need for costly empirical optimization and thus facilitate the protein design process. Successful computational design approaches range from analyzing phylogenetic data to infer useful structural, biophysical, and biochemical information to modeling the structure and function of proteins ab initio. The following chapter provides an overview over the theoretical considerations and experimental approaches that have been successful applied in the construction of synthetic protein switches.
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Extreme makeover yeast edition: de novo synthesis of five chromosomes | PLOS Synthetic Biology Community

Extreme makeover yeast edition: de novo synthesis of five chromosomes | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
– What I cannot create, I do not understand. This sentence taken from Richard Feynman’s board at the time of his death essentially captures, if applied to a
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Antibiotics v2.0: computational and synthetic biology approaches to combat antibiotic resistance

Antibiotics v2.0: computational and synthetic biology approaches to combat antibiotic resistance | SynBioFromLeukipposInstitute | Scoop.it
Antibiotics v2.0: computational and synthetic biology approaches to combat antibiotic resistance
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Application of Learning Classifier Systems to Gene Expression Analysis in Synthetic Biology

Learning classifier systems (LCS) are algorithms that incorporate genetic algorithms with reinforcement learning to produce adaptive systems described by if-then rules. As a new interdisciplinary branch of biology, synthetic biology pursues the design and construction of complex artificial biological systems from the bottom-up. A trend is growing in designing artificial metabolic pathways that show previously undescribed reactions produced by the assembly of enzymes from different sources in a single host. However, few researchers have succeeded thus far because of the difficulty in analyzing gene expression. To tackle this problem, data mining and knowledge discovery are essential. In this context, nature-inspired LCS are well suited to extracting knowledge from complex systems and thus can be exploited to investigate and utilize natural biological phenomena. This chapter focuses on applying LCS to gene expression analysis in synthetic biology. Specifically, it describes the optimization of artificial operon structure for the biosynthesis of metabolic pathway products in Escherichia coli. This optimization is achieved by manipulating the order of multiple genes within the artificial operons.
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Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10 µg ml−1, as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20–40 min following exposure to 3 µg ml−1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera.
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Bacteria Are Brewing Up the Next Generation of Antivenoms

Bacteria Are Brewing Up the Next Generation of Antivenoms | SynBioFromLeukipposInstitute | Scoop.it
DANIEL DEMPSEY WAS a grad student stationed in the jungles of Monteverde, Costa Rica when he first encountered the danger of a snakebite. The biologist was walking through the forest one day, catching bats to study them for malaria, when he almost stepped on the black, arrow-shaped head of an enormous pit viper—a fer-de-lance. That night as he described his encounter to the local family he was staying with, they began to tear up. They told him that earlier that year a “terciopelo,” what Costa Ricans call their country’s deadliest snake, had bitten the family’s five year-old niece. The hospital, a few hours drive away, didn’t have any antivenom in their stocks. She didn’t make it.

It was Dempsey’s memory of that little girl that made him leave his job as an antibody researcher at cancer pharmaceutical company, Celgene, to focus full-time on antivenoms. Every year, at least a hundred thousand people die from a run-in with one of the 375 venomous species of snake. And right now there’s a global shortage of the only thing that can save a bite victim: antivenom. For close to 100 years, antivenom production has been a laborious process of snake-milking and horse blood harvesting. But now, with synthetic biology and next-generation sequencing techniques, scientists are pushing the field into the future. Along with education and smart distribution, those advances could help end this global public health crisis.

A number of new biotech startups, both in the US and Europe, are signing on to tackle the antivenom problem. Dempsey’s, called Venomyx, is using vats of antibody-burping bacteria to engineer their cure. In a communal basement lab in San Francisco’s Tenderloin neighborhood, Dempsey and his employees Deepankar Roy and Alex Capovilla share bench, fridge, and instrument space to develop their pipeline of four antivenoms.

The first thing you might notice about the lab, if you know anything about antivenom production, is the distinct absence of farm animals. For decades, scientists have injected horses, or sometimes sheep, with a diluted version of snake venom, then collected their blood after a period of incubation and immune system triggering. Manufacturers use chemicals like ammonium sulfate or molecular separation methods to purify the antibodies. Then they suspend them in liquid and voila: antivenom. But to create their antibodies, Dempsey’s team is trading the equine incubator for the workhorse of the synthetic biology world: E. Coli, which they genetically modified to produce the venom-fighting stuff.

First, they injected a llama with sub-lethal amounts of snake venom. After sequencing the DNA of her antibody-creating B cells, they built a library of all the molecules those genes encoded. Then they exposed the library to tons of toxins found in snake venom—and after seeing which toxins stuck, they picked the tightest-binding molecules, and stuck their genes inside E. coli. The bacteria, when put in a bioreactor with the right mix of media and other molecules, kick out a soup of antibodies, which will go into different antivenoms for Asia, Africa, North America, and South America. Dempsey says that because llama antibodies are roughly 80 percent similar to the ones humans make, they don’t set off damaging immune responses, which can happen with horse and sheep-derived products.

But camelids aren’t the only new idea in town. A different startup, Copenhagen-based VenomAB, is instead using “humanized” antibodies to build out its antivenom product pipeline, which it has been developing with a Swedish pharmaceutical manufacturer. That process, which is popular in the cancer treatment world, involves designing human-like antibodies with variable regions that can bind different toxins, and getting bacteria to belch them out, just like they do for human insulin and other recombinant drug therapies. Both companies say that avoiding herds of hundreds or thousands of large mammals will bring down the time it takes to make the serums, as well as the cost. Typical doses can cost between $800 and $1,000 in rural Africa, and up to $14,000 in the US.

Solving the antivenom shortage, of course, is more complicated than simply developing more efficient production lines. The vast majority of mortalities fall on poor, isolated communities like subsistence farmers in Sub-Saharan Africa and Southeast Asia, so big manufacturers in the US and Europe have been slow to get into the game. In that vacuum, companies in Asia and India have flooded the market with subpar products, that might not even be applicable to places like Africa that have totally different species of snakes. The other big problem is that most of the antivenoms currently on the market require refrigeration. Developing countries, with their spotty infrastructure, needs shelf-stable serums that can survive tropical temperatures. “Good antivenoms can be made really affordably—$14 or $20 a vial,” says Leslie Boyer, founding director of the University of Arizona’s VIPER Institute. “But it’s the distribution costs that make the situation untenable.”

These new approaches to antivenom development are valuable from a research standpoint. But they may not be necessary to solve the global shortage. “Do we need the most cutting edge technologies modern science can offer?” says Boyer. “No. What we need is better distribution networks, and certification programs to regulate the quality of the products and education programs to build trust with communities.” To that end, she and her colleagues in Arizona are teaming up with experts from Mexico and Africa to launch an international awareness campaign to areas hardest hit by snakebites.

Dempsey is hoping that in a few years from now, when his shelf-stable, horse-free antivenoms are ready for prime time, the efforts of people like Boyer will make it easier to get treatments to people where they need it most. Places like the jungles of the Congo and the mountains of Costa Rica.
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Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics

Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics | SynBioFromLeukipposInstitute | Scoop.it
Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics.
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Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells

The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca2+). By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca2+-activated processes.
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Supramolecular Hydrogels Based on DNA Self-Assembly

Extracellular matrix (ECM) provides essential supports three dimensionally to the cells in living organs, including mechanical support and signal, nutrition, oxygen, and waste transportation. Thus, using hydrogels to mimic its function has attracted much attention in recent years, especially in tissue engineering, cell biology, and drug screening. However, a hydrogel system that can merit all parameters of the natural ECM is still a challenge. In the past decade, deoxyribonucleic acid (DNA) has arisen as an outstanding building material for the hydrogels, as it has unique properties compared to most synthetic or natural polymers, such as sequence designability, precise recognition, structural rigidity, and minimal toxicity. By simple attachment to polymers as a side chain, DNA has been widely used as cross-links in hydrogel preparation. The formed secondary structures could confer on the hydrogel designable responsiveness, such as response to temperature, pH, metal ions, proteins, DNA, RNA, and small signal molecules like ATP. Moreover, single or multiple DNA restriction enzyme sites could be incorporated into the hydrogels by sequence design and greatly expand the latitude of their responses. Compared with most supramolecular hydrogels, these DNA cross-linked hydrogels could be relatively strong and easily adjustable via sequence variation, but it is noteworthy that these hydrogels still have excellent thixotropic properties and could be easily injected through a needle. In addition, the quick formation of duplex has also enabled the multilayer three-dimensional injection printing of living cells with the hydrogel as matrix. When the matrix is built purely by DNA assembly structures, the hydrogel inherits all the previously described characteristics; however, the long persistence length of DNA structures excluded the small size meshes of the network and made the hydrogel permeable to nutrition for cell proliferation. This unique property greatly expands the cell viability in the three-dimensional matrix to several weeks and also provides an easy way to prepare interpenetrating double network materials. In this Account, we outline the stream of hydrogels based on DNA self-assembly and discuss the mechanism that brings outstanding properties to the materials. Unlike most reported hydrogel systems, the all-in-one character of the DNA hydrogel avoids the "cask effect" in the properties. We believe the hydrogel will greatly benefit cell behavior studies especially in the following aspects: (1) stem cell differentiation can be studied with solely tunable mechanical strength of the matrix; (2) the dynamic nature of the network can allow cell migration through the hydrogel, which will help to build a more realistic model to observe the migration of cancer cells in vivo; (3) combination with rapidly developing three-dimension printing technology, the hydrogel will boost the construction of three-dimensional tissues and artificial organs.
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First attempt to assemble full functional nitrogenase in eukaryotes

First attempt to assemble full functional nitrogenase in eukaryotes | SynBioFromLeukipposInstitute | Scoop.it
Centre for Plant Biotechnology and Genomics: R & D joint UPM-INIA centre - Centro de Biotecnología y Genómica de Plantas: centro mixto de I+D+i UPM-INIA.
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Synpromics Granted Key Patent in Synthetic Promoter Development

Synpromics Granted Key Patent in Synthetic Promoter Development | SynBioFromLeukipposInstitute | Scoop.it
Edinburgh, UK, 16th March 2017 / Sciad Newswire / Synpromics Ltd, the leading synthetic promoter and gene control company, is pleased to announce it has been granted a key patent (EP2668277B1) by the European Patent Office, protecting PromPT™ - its proprietary synthetic promoter development platform.
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Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light

In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.
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DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formation

Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.
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Scooped by Gerd Moe-Behrens
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Ultrasensitive Scaffold Dependent Protease Sensors with Large Dynamic Range

The rational construction of synthetic protein switches with pre-defined input-output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein-protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering five orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers and actuators.
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Scooped by Gerd Moe-Behrens
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What if Quantum Computers Used Hard Drives Made of DNA?

What if Quantum Computers Used Hard Drives Made of DNA? | SynBioFromLeukipposInstitute | Scoop.it
You can't save data on a quantum computer. So a commercial one will need to use vintage tech—ultra dense hard drives, maybe made of DNA or single atoms.
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Scooped by Gerd Moe-Behrens
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A resource dependent protein synthesis model for evaluating synthetic circuits

Reliable in silico design of synthetic gene networks necessitates novel approaches to model the process of protein synthesis under the influence of limited resources. We present such a novel protein synthesis model which originates from the Ribosome Flow Model and among other things describes the movement of RNA-polymerase and ribosomes on mRNA and DNA templates, respectively. By analyzing the convergence properties of this model based upon geometric considerations, we present additional insights into the dynamic mechanisms of the process of protein synthesis. Further, we demonstrate how this model can be used to evaluate the performance of synthetic gene circuits under different loading scenarios.
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