eLife - Open access to the most promising advances in science
Socrates Logos's insight:
by Owen W Ryan, Jeffrey M Skerker, Matthew J Maurer, Xin Li, Jordan C Tsai, Snigdha Poddar, Michael E Lee, Will DeLoache, John E Dueber, Adam P Arkin, Jamie H D Cate
"The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function."
"Cells proliferate by division into similar daughter cells, a process that lies at the heart of cell biology. Extensive research on cell division has led to the identification of the many components and control elements of the molecular machinery underlying cellular division. Here we provide a brief review of prokaryotic and eukaryotic cell division and emphasize how new approaches such as systems and synthetic biology can provide valuable new insight."
"With the recent dawn of synthetic biology, the old idea of man-made artificial life has gained renewed interest. In the context of a bottom-up approach, this entails the de novo construction of synthetic cells that can autonomously sustain themselves and proliferate. Reproduction of a synthetic cell involves the synthesis of its inner content, replication of its information module, and growth and division of its shell. Theoretical and experimental analysis of natural cells shows that, whereas the core synthesis machinery of the information module is highly conserved, a wide range of solutions have been realized in order to accomplish division. It is therefore to be expected that there are multiple ways to engineer division of synthetic cells. Here we survey the field and review potential routes that can be explored to accomplish the division of bottom-up designed synthetic cells. We cover a range of complexities from simple abiotic mechanisms involving splitting of lipid-membrane-encapsulated vesicles due to physical or chemical principles, to potential division mechanisms of synthetic cells that are based on prokaryotic division machineries."
" There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods."
by Eyal Karzbrun, Alexandra M. Tayar, Vincent Noireaux, Roy H. Bar-Ziv
"The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell."
"In 2011, Emmanuel Nnaemeka Nnadi needed help to sequence some drug-resistant fungal pathogens. A PhD student studying microbiology in Nigeria, he did not have the expertise and equipment he needed. So he turned to ResearchGate, a free social-networking site for academics, and fired off a few e-mails. When he got a reply from Italian geneticist Orazio Romeo, an international collaboration was born. Over the past three years, the two scientists have worked together on fungal infections in Africa, with Nnadi, now at Plateau State University in Bokkos, shipping his samples to Romeo at the University of Messina for analysis. “It has been a fruitful relationship,” says Nnadi — and they have never even met.
Ijad Madisch, a Berlin-based former physician and virologist, tells this story as just one example of the successes of ResearchGate, which he founded with two friends six years ago. Essentially a scholarly version of Facebook or LinkedIn, the site gives members a place to create profile pages, share papers, track views and downloads, and discuss research. Nnadi has uploaded all his papers to the site, for instance, and Romeo uses it to keep in touch with hundreds of scientists, some of whom helped him to assemble his first fungal genome.
More than 4.5 million researchers have signed up for ResearchGate, and another 10,000 arrive every day, says Madisch. That is a pittance compared with Facebook’s 1.3 billion active users, but astonishing for a network that only researchers can join. And Madisch has grand goals for the site: he hopes that it will become a key venue for scientists wanting to engage in collaborative discussion, peer review papers, share negative results that might never otherwise be published, and even upload raw data sets. “With ResearchGate we’re changing science in a way that’s not entirely foreseeable,” he says, telling investors and the media that his aim for the site is to win a Nobel prize....."
"A project begun some 13 years ago by Jay Keasling, the Associate Laboratory Director for Biosciences at Berkeley Lab and the CEO of the Joint BioEnergy Institute (JBEI), was culminated with an announcement on August 12 from the partnership of Sanofi, the multinational pharmaceutical company, and PATH, the nonprofit global health organization. Sanofi/PATH announced the shipment of 1.7 million treatments of semi-synthetic artemisinin to malaria-endemic countries in Africa. Unlike conventional artemisinin, which is derived from the bark of the sweet wormwood plant, this synthetic version of the World Health Organization’s frontline antimalarial drug is derived from yeast. The addition of a microbial-based source of artemisinin to the botanical source provides a stable new option for treating the millions of victims who are stricken with malaria each year, most of them children..."
"Silicon is so passé. Those who are truly au courant in the coding world are working with carbon—specifically DNA, that most ancient and elegant of codes. Such biohacking is central to the rapidly expanding field of synthetic biology, a term that somehow seems a little threatening to many of us who are the products of the old fashioned kind of biology that’s been around since the planet first managed to gin up a few primitive prokaryotes 3.5 billion years ago.
That’s especially because messing around with DNA is increasingly a garage band kind of venture. The basic techniques and technology are now sufficiently disseminated so that any reasonably bright and inquisitive person can do all kinds of interesting things in a home or community lab. And—gulp—might that not include weaponizing Ebola or involve some other highly anti-social endeavor?
Relax (really). Those fears are overblown, opines Nina DiPrimio, the editor of BioCoder, a quarterly published by and for the DIYbio (as in, Do It Yourself biology) community.
In the first place, says DiPrimio, endowing viruses with new and ever-more-lethal characteristics requires the kind of equipment and skill sets usually found only in large government or corporate labs. Second, if anyone wants to attempt it, the mischief-maker wouldn’t need to figure out how to manipulate Ebola or HIV. Relatively simple procedures already are known for weaponizing basic old anthrax, or manufacturing and distributing astoundingly powerful poisons such as ricin, or—well, you get the idea.
“It takes a lot of skill and equipment to do bad things” with gene-spliced microbes says DiPrimio, until recently a postdoctoral researcher at UC Berkeley and the co-organizer of the university’s Synthetic Biology Super Group. “A DNA cloning class won’t teach you how to create something pathogenic. That isn’t to say potential dangers should be ignored. At a certain point, regulation (of homegrown biohacking) is likely. We don’t know what that will look like, but the community is aware of it and discussing it.”
DiPrimio is also deeply concerned about safety within these do-it-yourself operations. BioCoder’s articles include pointers to ensure one’s lab is legal, and emphasize basic protocols so tyro researchers don’t burn, blow up or electrocute themselves. But first and foremost, the publication serves as an agora for the biohacking community...."
"There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods."
"The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication."
"Synthetic biology uses our understanding of biological systems to develop innovative solutions for challenges in fields as diverse as genetic control and logic devices, bioremediation, materials production or diagnostics and therapy in medicine by designing new biological components. RNA-based elements are key components of these engineered systems. Their structural and functional diversity is ideal for generating regulatory riboswitches that react with many different types of output to molecular and environmental signals. Recent advances have added new sensor and output domains to the existing toolbox, and demonstrated the portability of riboswitches to many different organisms. Improvements in riboswitch design and screens for selecting in vivo active switches provide the means to isolate riboswitches with regulatory properties more like their natural counterparts."
"IN THE SUMMER of 2009, a team of Cambridge University undergraduates built seven strains of the bacterium Escherichia coli, one in each color of the rainbow. Red and orange carotenoid pigments were produced by inserting genes from plant pathogen Pantoea ananatis; a cluster of genes from Chromobacterium violaceum were likewise modified to yield green and purple. The students’ technicolor creations, dubbed “E. chromi” in reference to the organisms’ scientific name, won the Cambridge team the grand prize at that year’s International Genetically Engineered Machines (iGEM) competition, in which high-school and college students engineer biology.
The students’ goals were not merely chromatic. Instead, they were building parts for biological machines. They engineered the genes into standardized forms called BioBricks: pieces of DNA that, like genetic Legos, are designed to be mixed and matched at will. Several thousand of these BioBricks, fulfilling various functions, are already housed in the MIT-based Registry of Standard Biological Parts. Some BioBricks detect chemicals like arsenic; others act as “tuners” that determine the threshold level of chemical input needed to turn on a certain gene. By combining the new color-producing genes with existing parts, the thinking went, one might easily construct biosensors that, in the presence of environmental toxins, produce output visible to the naked eye.
“E. chromi” struck a chord with designers Alexandra Daisy Ginsberg, G ’06, and James King, who began a collaboration with the iGEM team. In a short video that was named best documentary at the Bio:Fiction synthetic biology film festival in 2011, Ginsberg and King imagined possible futures for living color. Soon, they suggested, scientists might search the natural world for new biological pigments and the genes responsible, revolutionizing dye production. “E. chromi” in probiotic yogurt might monitor human disease while traveling through the gut; microbes in the atmosphere might change color to indicate air quality.
“I think it’s a new term to most of the public, synthetic biology,” mused the host of National Public Radio’s Science Friday in the fall of 2009 when he interviewed the Cambridge team. “But I guess we’re going to be hearing a lot more of it.”
How to Build a Biological Machine
ARMED with powerful new genetic tools and a penchant for tinkering, synthetic biologists have built a new menagerie. Photographic “E. coliroid” darken in response to light. Sensor bacteria record the presence of a chemical in a mouse’s gut by turning on certain genes. There are strains of E. coli that count input signals and others that carry out logical operations—steps toward biological computers. Still other strains smell like wintergreen and bananas instead of like the human gut. In 2005, festive researchers “wrote” the first verse of Viktor Rydberg’s Christmas poem “Tomten” into the genome of yet another E. coli strain, using triplets of DNA nucleotides to represent each letter; the resulting bacterium, they wrote, was “the first example of an organism that ‘recites’ poetry.”
Insofar as a common theme unites these diverse creations, it is the transformation of biology into an engineering discipline. Traditional genetic engineering amounted more or less to biological cut-and-paste: scientists could, for instance, transfer a cold-tolerance gene from an Arctic fish into a tomato. Synthetic biology aims for a more radical reorganization. Its organisms are built to be biological machines, with DNA and proteins standing in for circuit components or lines of computer code. In combination, the biological parts perform functions unknown to nature: processing signals, producing new chemicals, storing information.
“I like to say that biological carbon is the silicon of this century,” says Pamela A. Silver, Adams professor of biochemistry and systems biology at Harvard Medical School (HMS; see “Biology in This Century,” September-October 2011, page 72). Just as computers revolutionized the past hundred years, she says, biology is poised to transform the next. “The building of biological machines and biological computers—all of that should soon become a reality.”
To a certain mind, a cell already resembles a tiny, complex machine. It takes in chemicals from the environment and performs reactions to build new biological parts; it monitors signals and turns genes on and off in response. Cells have been compared to computers, to factories, to automatons. For a synthetic biologist with such complex systems already at hand, the task is to identify and manipulate the appropriate parts. “Many of the biomolecular components we’re not building from scratch,” says James J. Collins, Warren Distinguished Professor at Boston University and founding core faculty member at Harvard’s Wyss Institute for Biologically Inspired Engineering. “We’re taking native systems and then modifying them.”
Understanding and manipulating this elaborate machinery is a tough job. “I think of it as if some alien intelligence just dropped onto us all their intellectual property without documentation,” says George Church, Winthrop professor of genetics at HMS (see “DNA as Data,” January-February 2004, page 44). There’s no direct biological equivalent of a capacitor or the delete command, and synthetic biologists must creatively recombine existing biological parts in order to build new functions.
Take, for instance, the toggle switch, one of the simplest circuit components. A nonbiological example would be a light switch: it can be flipped between two discrete states, on or off, with nothing in between. In an abstract sense, the toggle switch amounts to a kind of memory, with its two states tantamount to 0’s and 1’s. Such bistability has some analogues in nature. Venus fly traps, for instance, have structures that alternate between open and shut (see “Leaves That Lunch,” May-June 2005, page 14). Specific signals instruct cells whether to remain dormant or divide. Some viruses also toggle between two distinct states of dormancy or active infection.
When Collins’s lab built a bacterial toggle switch—one of the first pieces of biological circuitry—they made it from two genes. Each encoded a repressor protein for the opposite gene; once one gene was turned on, it turned the other gene off. The switch could be flipped by giving the cell a specific chemical signal, disabling the active repressor protein and allowing the other to take hold. With the second gene now turned on, turning off the first, the switch would stay flipped long after the signal had disappeared. “As a cellular memory unit,” wrote Collins when his team published its design in 2000, “the toggle forms the basis for ‘genetic applets’—self-contained, programmable, synthetic gene circuits for the control of cell function.”
Genetic applets (perhaps more aptly, apps today) are one of synthetic biology’s defining goals. Some 40 years after scientists began learning to rearrange DNA, genetic engineering remains something of a cottage industry. In a time-consuming, almost artisanal craft, researchers modify organisms ad hoc to suit their particular needs. Synthetic biology was born out of a desire for greater, more versatile control, says Silver, who took part in early meetings of the Synthetic Biology Working Group at MIT. “The question that forms the core of synthetic biology is, ‘Why can’t biology be easier and more predictable to engineer?’ ”
Indeed, for synthetic biologists, it is not enough to have painstakingly built genetic switches and biological machines. “Right now, people—especially graduate students—just spend an inordinate amount of time making DNA and figuring out how to put DNA together,” says Jeffrey Way, senior staff scientist at the Wyss Institute, who is married to Silver. “It’s extremely time-consuming.”
"Scientists have made an amplifier to boost biological signals, using DNA and harmless E. coli bacteria.
Conventional amplifiers, such as those that are combined with loudspeakers to boost the volume of electric guitars and other instruments, are used to increase the amplitude of electrical signals.
Now scientists from Imperial College London have used the same engineering principles to create a biological amplifier, by re-coding the DNA in the harmless gut bacteria Escherichia coli bacteria (E. coli).
The team say this ‘bio-amplifier’ might be used in microscopic cellular sensors , which scientists have already developed, that could detect minute traces of chemicals and toxins, to make them more sensitive. Ultimately, this could lead to new types of sensors to detect harmful toxins or diseases in our bodies and in the environment before they do any damage.
In laboratory tests, the team’s bio-amplifier was able to significantly boost the detection limit and sensitivity of a sensor designed to detect the toxin arsenic. The device is also modular, which means that the devices can be easily introduced in different genetic networks, and can potentially be used to increase the sensitivity and accuracy of a broad range of other genetic sensors to detect pathogens and toxins.
The results of the study are published in the journal Nucleic Acids Research.
Dr Baojun Wang, who is now based at the University of Edinburgh, but carried out the study while in the Division of Cell and Molecular Biology at Imperial, said: “One potential use of this technology would be to deploy microscopic sensors equipped with our bio-amplifier component into a water network. Swarms of the sensors could then detect harmful or dangerous toxins that might be hazardous to our health. The bio-amplifiers in the sensors enable us to detect even minute amounts of dangerous toxins, which would be of huge benefit to water quality controllers.”
Scientists have previously known that cells have their own inbuilt amplifiers to first detect and then boost biological signals, which are crucial for survival and reproduction. They have been attempting to understand how they work in more detail so as to remodel them for other applications. However the challenge for scientists has been engineering a device that can predictably amplify signals without distortion or feedback.
In the study, scientists first re-engineered genes involved in a special cell network called hrp (hypersensitive response and pathogenicity), which have naturally occurring amplifying proteins that function just like an electronic amplifier. They then cloned these amplifying components and inserted them into the harmless gut bacteria E. coli, fitting it with a synthetic arsenic input sensor and a fluorescent green protein gene as the output. ..."
Please share! Diego should not go to jail for this.
*Diego Gomez is facing four to eight years in prison for sharing an academic article on the Internet*
BY MAIRA SUTTON "In many parts of the developing world, students face barriers to access academic materials. Libraries are often inadequate, and schools and universities are often unable to pay dues for expensive, specialized databases. For these students, the Internet is a vital tool and resource to access materials that are otherwise unavailable to them. Yet despite the opportunities enabled by the Internet, there are still major risks to accessing and sharing academic resources online.
A current situation in Colombia exemplifies this problem: a graduate student is facing four to eight years in prison for sharing an academic article on the Internet. He wasn't making a personal profit from sharing the article—he simply intended for other scientists like him to be able to access and cite this scientific research.
Diego Gomez, 26, is a Master's student who has been researching biodiversity and working on the conservation of reptiles and amphibians for several years in the South American region. Throughout his young career, the biggest obstacle he faced was in accessing academic resources that existed on global research databases. As a student at a small university in Armenia, the availability of research papers was so limited that he often had to save money to make trips to Bogotá to access biological collections, articles, and databases only available to him at natural history museums and libraries at the capital city.
Over time, he increasingly came to depend on the Internet. It enabled him to read relevant research, share documents, and communicate with others in his field. Despite the online resources that were available, there were still major barriers that prevented him from accessing the plethora of research that existed. So when he and others came across papers that were crucial to their work, they often shared it online for other researchers to access.
The important thing is to make a correct citation, attributing researchers’ work by indicating their name and year of publication and, of course, not claiming the work of another researcher, but to recognize it and value it. Therefore, what we usually do is to reference the findings and make them available to those who need them.
One day a couple of years ago, he came across a paper that was especially useful to his field work. He then later shared the research online on the site, Scribd. The author of the paper then filed a lawsuit over the “violation of [his] economic and related rights.” Under the allegations of this lawsuit, Gomez could be sent to prison for up to eight years and face crippling monetary fines.
The Criminal Charges
He is being sued under a criminal law that was reformed in 2006, following the conclusion of a free trade agreement between Colombia and the United States. The new law was meant to fulfill the trade agreement's restrictive copyright standards, and it expanded criminal penalties for copyright infringement, increasing possible prison sentences and monetary fines.
Colombian digital rights organization, Fundación Karisma, is supporting Gomez in his case to fight against these excessive criminal charges. Carolina Botero, staff attorney at Fundación Karisma writes (translated from Spanish):
The rationale is the potential damage that "piracy" in the industry generates. Without prejudice to the pending debate on the subject, it should be clear that the actions of users, non-profit activities, and sharing, are not crimes. […] In a society that has a disruptive technology like the Internet, the exercise of the rights to education, access to science and culture, and respect for freedom of expression must be respected.
Colombia does not have flexible fair use system like in the United States. It has a closed list of exceptions and limitations to the rights of authors (derecho de autor). This list was issued more than 20 years ago and are narrowly tailored to some specific situations that are not at all applicable to the digital age. Therefore none of these will apply directly to his case even if it was done for educational purposes.
There is a Supreme Court ruling that further weighs this legal consideration in his favor. In 2008, the highest Colombian court ruled that an infringing activity can only be criminal if there was intention to profit from the copyrighted work. The decision was partially based on international law—the Berne Convention, which carries an exceptions and limitations framework called the three-step test. This test is a way of determining whether a certain use is legal as long as it doesn't conflict with the “normal exploitation of the work and does not unreasonably prejudice the legitimate interests of the author.” Since Gomez was clearly not sharing academic articles for personal profit, there is firm ground to assert that his actions were not criminal. Botero of Fundación Karisma comments on this point:
In 2011, Diego published on the Internet a thesis that was defended in 2006. The fact that a scholar author believes that after 5 years someone who spreads his scientific findings is harming his economic interests totally ignores the importance of science in development, in this case, in the conservation of the biodiversity in Colombia, the second most biologically diverse country in the world.
This case exemplifies the real life harm of overreaching restrictions due to excessive laws that protect the “economic rights” of authors. Gomez only wanted to share these articles to further his life mission to protect native wildlife and to allow others with a similar passion to access this research. He is only one of countless thousands who risk themselves every day to push against the prohibitive restraints of copyright. We need major reform of our laws, both internationally and domestically, to ensure that people are not made criminals for promoting scientific progress and exercising their creative expression. In other words, for doing exactly what authors' rights laws are allegedly intended to do."