Yoshiyuki Shibata, Pankaj Kumar1, Ryan Layer, Smaranda Willcox, Jeffrey R. Gagan, Jack D. Griffith, Anindya Dutta
We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5′-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the “Thousand Genomes” project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci."
A biobrick library for cloning custom eukaryotic plasmids
by Marco Constante, Raik Grünberg, Mark Isalan
"Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/)." http://bit.ly/IJAFT3
Li NS, Frederiksen JK, Piccirilli JA. "RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3'- or 5'-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage. Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3'- or 5'-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3'- or 5'-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3'- and 5'-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities." http://1.usa.gov/IN2DK0
Prepared by the Lab’s Synthetic Biology Working Group, formed under the direction of Sam Chapman, manager of State & Community Relations and including representatives from Public Affairs, Federal Government Relations, the Joint Genome Institute, Joint BioEnergy Institute, Environmental Health & Safety, UC Berkeley and others.
What is synthetic biology?
Synthetic biology combines modern principles of science and engineering to develop novel biological functions and systems.
This includes the design and construction of new biological devices, such as molecules, genetic circuits or cells, and the re-designing and engineering of existing biological systems, such as microorganisms. The goal is to engineer microorganisms to produce valuable chemical products from simple, inexpensive and renewable starting materials, such as sugars, in a sustainable manner.
Is synthetic biology an entirely new and untested field of science?
No. Through selective breeding, humans have been altering the genetic code of plants and animals for more than a thousand years. Synthetic biology today is an extension of the genetic engineering research that began about 40 years ago. Also known as recombinant DNA technology, genetic engineering has produced valuable chemical products such as human insulin and growth hormones, hormones for treating infertility, monoclonal antibodies and the hepatitis B vaccine. Through better understanding of biological systems and metabolic pathways, coupled with enormous advances in computing, synthetic biology moves beyond these earlier efforts to make the design and production of biological parts and systems more predictable and reliable. Berkeley Lab scientists will apply synthetic biology to the production of advanced biofuels that can replace gasoline, diesel and jet fuel in today’s engines and can be transported via existing pipeline infrastructures. Spinoffs from this research will include medical drugs, biodegradable and green chemistry replacements for other petrochemical products, such as plastics, and environmental restoration through decontamination of hazardous pollutants.
How safe is synthetic biology?
Out of four potential biosafety levels (BLs) of containment detailed in national and international standards, (BL1=least dangerous to BL4) Berkeley Lab operates at only the two lowest and most common biosafety levels, BL1 and BL2.
Synthetic biology research at JBEI typically operates at the BL1 level, which is defined as being: “Suitable for work involving agents of unknown or minimal potential hazard to laboratory personnel and the environment, or work with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans.”
Through the Integrated Safety Management (ISM) program the Lab meets or exceeds standards for biosafety, worker safety and health, and environmental protection set by the National Institutes of Health (NIH) and Centers for Disease Control and Prevention (CDC) for the biosafety of recombinant and other research; California Department of Public Health (CDPH) for biohazardous waste; Occupational Safety and Health Administration (OSHA) for bloodborne pathogens; U.S. Department of Agriculture (USDA) for protection of animal and plant resources; and other agencies.
Biosafety of research is also reviewed by a Lab Institutional Biosafety Committee (IBC) comprised of peer researchers, safety and health experts, and members of the public. IBC reviews include assessment and documentation of any risks and required controls.
Berkeley Lab maintains a robust incident and emergency reporting and response program in which staff and management are trained and encouraged to report incidents. The California Department of Public Health inspects the Lab for compliance with medical waste storage and disposal requirements and has consistently found the Lab’s program in compliance.
Cramming biology on silicon is not a new effort, and it’s one that has helped advance the science of genomics and led to the $1,000 genome. But in the last few weeks, I’ve noticed some pretty sweet combinations of biology and chip research. They will first end up helping biological research and test new drug compounds, but in the future might become something out of Ray Kurzweil’s The Singularity, the melding of man and machine.
A gut chip for a gut check: You and I might not want a chip that mimics the entire process of a human digestive system including peristalsis and living bacteria, but folks trying to test out drugs do. And now, thanks to researchers at Harvard University they have one. The lab-on-chip uses two thin channels coated in a biological growth medium and human intestinal cells to create a mini-intestine that apparently can sustain actual gut bacteria for about a week. Who needs an ant farm anymore?...
This is awesome. Launching today is Microryza, a Seattle-based firm that is taking the popular crowd-based fundraising model and is applying it to a critical component of the modern economy: scientific research.
Yes, you just might be able to help fund your own Sheldon Cooper now. The platform allows for direct donation to researchers that are working on the things that you care about, allowing you to put your money where your nerdy hunches and passions are. I would fund people looking to figure out if there was ever life on Mars. That’s just me.
Rafael Silva-Rocha, Victor de Lorenzo "Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods." http://bit.ly/HzC5My
"The Internet is a fascinating appliance. The ultimate in far-flung peer-to-peer systems, it allows consumers to double as producers and distributers of content. It's a revolution on par with widespread literacy and the printing press. But the same media that topple dictators and expose wrongdoing can also be used to spread fear, uncertainty, and doubt (FUD).
Panic Feeds on FUD
FUD—the term—gained popularity in the early days of open-source desktop computing, when companies concerned with their marketshare would spread vague rumors of infringements or security holes in competing free software tools. Ruled by its users, the Internet was partitioned into circles of common interest; sources with a particular bias could be counted on to link to material supporting their viewpoints, and that material was often hosted by a user of a similar persuasion. Free as always, the answers were still out there; but the deluge of information can be daunting when many stories are retweets or reposts or links and so on.
What's so wrong with that? Why not let users hang out in their personal echo chambers? If they want the other side of the story, there's always Google. However, Google does not think for you—you need to know the right questions to ask it, and you need the ability to pick out the most relevant (and reliable) pieces of data. It does not discriminate between misinformation and fact, so FUD can hang around for decades, long after it has been thoroughly debunked. It is repeated in these echo chambers until it is dogma and serves to prop up extreme positions.
There are precious few occupations that do not draw the ire of some fringe group—firefighters, perhaps. Actually, amend that—volunteer firefighters; as some sites claim the municipal ones are overpaid. Well, you say tomato, I say inferno.
So it really comes as no surprise that there are echo chambers devoted to covering synthetic biology misdeeds. It's the same groups who have protested GMOs for decades now, and their perennial reports on the dangers of genetic engineering are akin to the phonebooks once regularly delivered to every doorstep...."
Képès F, Jester BC, Lepage T, Rafiei N, Rosu B, Junier I.
"Recently the mismatch between our newly acquired capacity to synthetize DNA at genome scale, and our low capacity to design ab initio a functional genome has become conspicuous. This essay gathers a variety of constraints that globally shape natural genomes, with a focus on eubacteria. These constraints originate from chromosome replication (leading/lagging strand asymmetry; gene dosage gradient from origin to terminus; collisions with the transcription complexes), from biased codon usage, from noise control in gene expression, and from genome layout for co-functional genes. On the basis of this analysis, lessons are drawn for full genome design..."
"The time may soon come for chemists to throw away all their old glass reaction vessels. UK and Norwegian scientists have now shown that far better and more effective vessels can be fabricated on a 3D printer.
Not only do 3D printers offer the possibility of producing vessels with much more complex architectures, but the vessels can be designed to influence the course of the reaction or even to take part in it. And if that's not enough, 3D printers also offer a convenient way to introduce reactant chemicals into the vessels.
'I have always been interested in new ways of doing complex chemistry and exploring new reaction spaces,' explains lead researcher Lee Cronin at the University of Glasgow. 'I had never used a 3D printer before but met a researcher interested in designing new objects for 3D printed architecture and got to asking them about combining it with chemistry.'..."
Shahar Alon, Eyal Mor, Francois Vigneault, George Church, Franco Locatelli, Federica Galeano, Angela Gallo, Noam Shomron and Eli Eisenberg
"A-to-I editing modifies RNA transcripts from their genomic blueprint. A prerequisite for this process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Editing of miRNAs has the potential to add another layer of complexity to gene regulation pathways, especially if editing occurs within the miRNA-mRNA recognition site. Thus, it is of interest to study the extent of this phenomenon. Current reports in the literature disagree on its extent; while some reports claim that it may be widespread others deem the reported events as rare. Utilizing a next-generation sequencing (NGS) approach supplemented by an extensive bioinformatic analysis, we were able to systematically identify A-to-I editing events in mature miRNAs derived from human brain tissues. Our algorithm successfully identified many of the known editing sites in mature miRNAs, and revealed 17 novel human sites, 12 of which are in the recognition sites of the miRNAs. We confirmed most of the editing events using in-vitro ADAR over-expression assays. The editing efficiency of most sites identified is very low. Similar results are obtained for publicly available datasets of mouse brain-regions tissues. Thus, we find that A-to-I editing does alter several miRNAs but it is not widespread." http://bit.ly/ITHN0m
"In a recent Penny Stock Detectives article, editor Danny Esposito reports on opportunities in synthetic biology, a revolutionary biotechnology that engineers and so rebuilds natural systems, imitating nature. Esposito reveals that there are biotech companies creating an artificial gene, with the only function of absorbing oil that has been spilled in the ocean, or creating a synthetic gene with the sole purpose of absorbing the harmful pollution that is emitted right from the manufacturer’s site. According to the editor, these are just few examples of the profits that can be made with synthetic biology."
A Method for Fast, High-Precision Characterization of Synthetic Biology Devices
by Jacob Beal1, Ron Weiss2, Fusun Yaman1, Noah Davidsohn2, and Aaron Adler1 1Raytheon BBN Technologies and 2MIT
"Engineering biological systems with predictable behavior is a founda- tional goal of synthetic biology. To accomplish this, it is important to accurately characterize the behavior of biological devices. Prior charac- terization efforts, however, have generally not yielded enough high-quality information to enable compositional design. In the TASBE (A Tool-Chain to Accelerate Synthetic Biological Engineering) project we have developed a new characterization technique capable of producing such data. This document describes the techniques we have developed, along with exam- ples of their application, so that the techniques can be accurately used by others."
Posted by Thomas Howard One of the advantages of working with bacteria is that genes can be clustered together under control of a single promoter (known as an operon). Multiple protein products can therefore be generated from a single messenger RNA strand (the mRNA is said to be polycistronic (Fig. 1)). As a result entire metabolic pathways can be created using a single promoter.
In eukaryotic systems this is generally not the case. mRNA in eukaryotes is typically monocistronic (there are a few exceptions but these undergo post-transcriptional processing and are not viewed as truly polycistronic e.g. lower box Fig. 1). This limitation means more DNA cloning/synthesis, repeated rounds of transformation and increased difficulty in controlling the expression levels of different genes – which can be important for metabolic engineering, for example.
Massimiliano Zampini and Finbarr Hayes "Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon–helix–helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel β-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common α-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the α-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea."
see also: DIGITAL PICK: ELEMENTS OF SURPRISE Posted by The New Yorker "In 2009, Michael Specter wrote about synthetic biology and the stunningly complex knowledge of basic elements that the field requires. “[Scientists] are attempting to reconfigure the metabolic pathways of cells to perform entirely new functions, such as manufacturing chemicals and drugs,” he explained. “Eventually, they intend to construct genes—and new forms of life—from scratch.” Budding synthetic biologists—and average folks whose knowledge of the periodic table stopped with sophomore-year chemistry—would be wise to check out NOVA Elements, a new app from PBS that explains why the periodic table has its specific shape, what gives each element its properties, and how the elements combine to do what they do. The app also offers “Essential Elements,” an element-building game hosted by David Pogue, of the Times, and a two-hour NOVA video, “Hunting the Elements.”" http://nyr.kr/HAozg0
"It began with a frustrated blogpost by a distinguished mathematician. Tim Gowers and his colleagues had been grumbling among themselves for several years about the rising costs of academic journals.
They, like many other academics, were upset that the work produced by their peers, and funded largely by taxpayers, sat behind the paywalls of private publishing houses that charged UK universities hundreds of millions of pounds a year for the privilege of access.
There had been talk last year that a major scientific body might come out in public to highlight the problem and rally scientists to speak out against the publishing companies, but nothing was happening fast.
So, in January this year, Gowers wrote an article on his blog declaring that he would henceforth decline to submit to or review papers for any academic journal published by Elsevier, the largest publisher of scientific journals in the world.
He was not expecting what happened next. Thousands of people read the post and hundreds left supportive comments. Within a day, one of his readers had set up a website, The Cost of Knowledge, which allowed academics to register their protest against Elsevier.
The site now has almost 9,000 signatories, all of whom have committed themselves to refuse to either peer review, submit to or undertake editorial work for Elsevier journals. "I wasn't expecting it to make such a splash," says Gowers. "At first I was taken aback by how quickly this thing blew up."...."
Caleb Garling "What do you do with a 200-terabyte instruction manual that tells you how to build a human? You put it on a cloud. That’s what Amazon and the National Institute of Health (NIH) have done with the 1000Genomes project, using Amazon’s S3 storage service to offer over 1,700 human genomes to genetics researchers across the globe. “This is what allows us to drive more complex maps of how genes interact with each other and their environment and zoom in on areas that may have a role to play in human health and disease,” says Matt Wood, who oversees Amazon’s side of the project and holds a PhD in bioinformatics. “This is the seed to create a tree of data.” Amazon and the NIH made a big splash last month when they announced that anyone with an S3 account could now access this data, but the move is only part of a much larger effort to reinvent genetics using the proverbial cloud, with researchers tapping into public services from the likes of Amazon, Google, and Microsoft but also building their own cloud services using tools such as Hadoop, the open source platform for crunching large amounts of data across a sea of ordinary servers...." http://bit.ly/HKcOBF