This video is a collaboration with the University of Manchester iGEM Team. Here we show a basic tutorial on how to mathematically model biological systems .
One remarkable achievement in synthetic biology was the re-construction of mycoplasma genomes and their cloning in yeast where they can be modified using available genetic tools. Recently, CRISPR/Cas9 editing tools were developed for yeast mutagenesis. Here, we report their adaptation for the engineering of bacterial genomes cloned in yeast. A seamless deletion of the mycoplasma glycerol-3-phosphate oxidase-encoding gene (glpO) was achieved without selection in one step, using 90 nt paired oligonucleotides as templates to drive recombination. Screening of the resulting clones revealed that more than 20% contained the desired deletion. After manipulation, the overall integrity of the cloned mycoplasma genome was verified by multiplex PCR and PFGE. Finally, the edited genome was back-transplanted into a mycoplasma recipient cell. In accordance with the deletion of glpO, the mutant mycoplasma was affected in the production of H2O2. This work paves the way to high-throughput manipulation of natural or synthetic genomes in yeast.
There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.
An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position-function relations were quantified for the terminator tR2 in Escherichia coli. The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE-distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology.
Joe Davis is an artist who works not only with paints or pastels, but also with genes and bacteria. In 1986, he collaborated with geneticist Dan Boyd to encode a symbol for life and femininity into an E. coli bacterium. The piece, called Microvenus, was the first artwork to use the tools and techniques of molecular biology. Since then, bioart has become one of several contemporary art forms (including reclamation art and nanoart) that apply scientific methods and technology to explore living systems as artistic subjects. A review of the field, published November 23, can be found in Trends in Biotechnology. Bioart ranges from bacterial manipulation to glowing rabbits, cellular sculptures, and—in the case of Australian-British artist Nina Sellars—documentation of an ear prosthetic that was implanted onto fellow artist Stelarc's arm. In the pursuit of creating art, practitioners have generated tools and techniques that have aided researchers, while sometimes crossing into controversy, such as by releasing invasive species into the environment, blurring the lines between art and modern biology, raising philosophical, societal, and environmental issues that challenge scientific thinking. "Most people don't know that bioart exists, but it can enable scientists to produce new ideas and give us opportunities to look differently at problems," says author Ali K. Yetisen, who works at Harvard Medical School and the Wellman Center for Photomedicine, Massachusetts General Hospital. "At the same time there's been a lot of ethical and safety concerns happening around bioart and artists who wanted to get involved in the past have made mistakes." The Evolution of Bioart In between experiments, Alexander Fleming would paint stick figures and landscapes on paper and in Petri dishes using bacteria. In 1928, after taking a brief hiatus from the lab, he noticed that portions of his "germ paintings," had been killed. The culprit was a fungus, penicillin—a discovery that would revolutionize medicine for decades to come. In 1938, photographer Edward Steichen used a chemical to genetically alter and produce interesting variations in flowering delphiniums. This chemical, colchicine, would later be used by horticulturalists to produce desirable mutations in crops and ornamental plants. In the late 18th and early 19th centuries, the arts and sciences moved away from traditionally shared interests and formed secular divisions that persisted well into the 20th century. "Appearance of environmental art in the 1970s brought about renewed awareness of special relationships between art and the natural world," Yetisen says.
Bioart is a creative practice that adapts scientific methods and draws inspiration from the philosophical, societal, and environmental implications of recombinant genetics, molecular biology, and biotechnology. Some bioartists foster interdisciplinary relationships that blur distinctions between art and science. Others emphasize critical responses to emerging trends in the life sciences. Since bioart can be combined with realistic views of scientific developments, it may help inform the public about science. Artistic responses to biotechnology also integrate cultural commentary resembling political activism. Art is not only about ‘responses’, however. Bioart can also initiate new science and engineering concepts, foster openness to collaboration and increasing scientific literacy, and help to form the basis of artists’ future relationships with the communities of biology and the life sciences.
Rachel Armstrong talks about innovating and designing sustainable habitable structures that use new technologies such as synthetic biology and smart ...
MIT researchers have developed a biomedical imaging system that could ultimately replace a $100,000 piece of a lab equipment with components that cost just hundreds of dollars. The system uses a technique called fluorescence lifetime imaging, which has applications in DNA sequencing and cancer diagnosis, among other things. So the new work could have implications for both biological research and clinical practice. "The theme of our work is to take the electronic and optical precision of this big expensive microscope and replace it with sophistication in mathematical modeling," says Ayush Bhandari, a graduate student at the MIT Media Lab and one of the system's developers. "We show that you can use something in consumer imaging, like the Microsoft Kinect, to do bioimaging in much the same way that the microscope is doing." The MIT researchers reported the new work in the Nov. 20 issue of the journal Optica. Bhandari is the first author on the paper, and he's joined by associate professor of media arts and sciences Ramesh Raskar and Christopher Barsi, a former research scientist in Raskar's group who now teaches physics at the Commonwealth School in Boston. Fluorescence lifetime imaging, as its name implies, depends on fluorescence, or the tendency of materials known as fluorophores to absorb light and then re-emit it a short time later. For a given fluorophore, interactions with other chemicals will shorten the interval between the absorption and emission of light in a predictable way. Measuring that interval—the "lifetime" of the fluorescence—in a biological sample treated with a fluorescent dye can reveal information about the sample's chemical composition. In traditional fluorescence lifetime imaging, the imaging system emits a burst of light, much of which is absorbed by the sample, and then measures how long it takes for returning light particles, or photons, to strike an array of detectors. To make the measurement as precise as possible, the light bursts are extremely short. The fluorescence lifetimes pertinent to biomedical imaging are in the nanosecond range. So traditional fluorescence lifetime imaging uses light bursts that last just picoseconds, or thousandths of nanoseconds.
We review the rapidly emerging field of switchable interfaces and its implications for bioelectronics. We seek to piece together early breakthroughs and key developments, and highlight and discuss the future of switchable bioelectronics by focusing on bio-electrochemical processes based on mimicking and controlling biological environments with external stimuli. All these studies strive to answer a fundamental question: “how do living systems probe and respond to their surroundings? And, following on from that: “how one can transform these concepts to serve the practical world of bioelectronics?” The central obstacle to this vision is the absence of versatile interfaces that are able to control and regulate the means of communication between biological and electronic systems. Here, we review the overall progress made to date in building such interfaces at the level of individual biomolecules and focus on the latest efforts to generate device platforms that integrate bio-interfaces with electronics.
Decrypting Synthetic Biology: Engineering Microbes by Ms. Aileen Bayot-Custodio University Researcher II National Institute of Molecular Biology and ...
Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments1, 2, 3, 4, 5, 6. The universally conserved Cas1–Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7, 8, 9, 10, 11, 12, 13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2, 3, 4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
The cell cycle plays a key role in human health and disease, including development and cancer. The ability to easily and reversibly control the mammalian cell cycle could mean improved cellular reprogramming, better tools for studying cancer, more efficient gene therapy, and improved heterologous protein production for medical or industrial applications. RESULTS: We engineered RNA-based control devices to provide specific and modular control of gene expression in response to exogenous inputs in living cells. Specifically, we identified key regulatory nodes that arrest U2-OS cells in the G0/1 or G2/M phases of the cycle. We then optimized the most promising key regulators and showed that, when these optimized regulators are placed under the control of a ribozyme switch, we can inducibly and reversibly arrest up to ~80 % of a cellular population in a chosen phase of the cell cycle. Characterization of the reliability of the final cell cycle controllers revealed that the G0/1 control device functions reproducibly over multiple experiments over several weeks. CONCLUSIONS: To our knowledge, this is the first time synthetic RNA devices have been used to control the mammalian cell cycle. This RNA platform represents a general class of synthetic biology tools for modular, dynamic, and multi-output control over mammalian cells.
Synthetic gene networks have evolved from simple proof-of-concept circuits to complex therapy-oriented networks over the past 15 years. This advancement has greatly facilitated the expansion of the emerging field of synthetic biology. In this review, we highlight the main applications ofsynthetic gene networks in understanding biological design principles, developing biosensors for diagnosis, producing industrial and biomedical compounds, and treating human diseases. Finally, we outline current challenges and future prospects of synthetic gene networks for advancing practical applications.
Collaborating with designers, who sometimes take a highly conceptual approach, can help scientists to envisage exciting new ideas and applications. A good example of this is the work of Victoria Geaney, a designer who worked with synthetic biology students at Imperial College London to bring together the worlds of Fashion and Science. Victoria came to the University of Cambridge on the 30th of October to give a lecture, so we thought it would be a good opportunity to send Sarah Sewell to investigate:
The students in Anthony James’s basement insectary at the University of California, Irvine, knew they’d broken the laws of evolution when they looked at the mosquitoes’ eyes.
By rights, the bugs, born from fathers with fluorescent red eyes and mothers with normal ones, should have come out only about half red. Instead, as they counted them, first a few and then by the hundreds, they found 99 percent had glowing eyes.
More important than the eye color is that James’s mosquitoes also carry genes that stop the malaria parasite from growing. If these insects were ever released in the wild, their “selfish” genetic cargo would spread inexorably through mosquito populations, and potentially stop the transmission of malaria.
The technology, called a “gene drive,” was built using the gene-editing technology known as CRISPR and is being reported by James, a specialist in mosquito biology, and a half dozen colleagues today in the Proceedings of the National Academy of Sciences.
A functioning gene drive in mosquitoes has been anticipated for more than a decade by public health organizations as a revolutionary novel way to fight malaria. Now that it’s a reality, however, the work raises questions over whether the technology is safe enough to ever be released into the wild.
“This is a major advance because it shows that gene drives will likely be effective in mosquitoes,” says Kevin Esvelt, a gene drive researcher at Harvard University’s Wyss Institute. “Technology is no longer the limitation.”
Starting last summer, Esvelt and other scientists began warning that gene drives were about to jump from theory to reality (see “Protect Society from Our Inventions, Says Genome Editing Scientists”) and needed more attention by regulators and the public. The National Academy of Sciences is studying the science and ethics of the technology and plans to release recommendations next year on “responsible conduct” by scientists and companies.
Gene drives are just the latest example of the fantastic power of CRISPR editing to alter the DNA of living things, which has already set off a debate over the possibility that gene editing could be used to generate designer human babies (see “Engineering the Perfect Baby”). But Henry Greely, a law professor and bioethics specialist at Stanford, says environmental uses are more worrisome than a few modified people. “The possibility of remaking the biosphere is enormously significant, and a lot closer to realization,” he says.
Malaria is caused when a mosquito bite transmits plasmodium, a single-celled parasite. It’s treatable, yet every year, 670,000 people die from malaria, the majority of them young children in sub-Saharan Africa.
James says his mosquitoes are the culmination of decades of mostly obscure, unheralded work by a few insect specialists toward constructing a genetic solution to malaria. It finally became possible this year when scientists in the laboratory of Ethan Bier, a fly biologist at the University of California, San Diego, who is a coauthor of the paper, finally used CRISPR to perfect a molecular “motor” that could allow the anti-malaria genes to spread.
The mosquitoes have two important genetic additions. One is genes that manufacture antibodies whenever a female mosquito has a “blood meal.” Those antibodies bind to the parasite’s surface and halt its development. Yet normally, such an engineered mosquito would pass the genes only to exactly half its offspring, since there’s a 50 percent chance any chunk of DNA would come from its mate. And since the new genes probably don’t help a mosquito much, they’d quickly peter out in the wild.
That’s where CRISPR comes in. In a gene drive, components of the CRISPR system are added such that any normal gene gets edited and the genetic cargo is added to it as well. In James’s lab, practically all the mosquitoes ended up with the genetic addition, a result Esvelt calls “astounding.”
What worries Esvelt is that, in his opinion, the California researchers haven’t used strict enough safety measures. He says locked doors and closed cages aren’t enough. He wants them to install a genetic “reversal drive” so the change can be undone, if necessary. “An accidental release would be a disaster with potentially devastating consequences for public trust in science and especially gene-drive interventions,” he says. “No gene-drive intervention must ever be released without popular support.”
James says the experiment was safe since the mosquitoes are kept behind a series of locked, card-entry doors and because they aren’t native to California. If any escaped, they wouldn’t be able to reproduce.
In fact, the whole point of a gene drive is to release it into the wild, a concept that has long been accepted, at least in theory, by public health organizations including the Gates Foundation. Now that they’re actually possible, however, alarming news headlines have compared the technology to “the next weapon of mass destruction” and even raised the specter of insect terrorism, such as mosquitoes that kill people with a toxin.
Gene-drive terrorism is probably nonsense, at least for now. That’s because even if insect weapons were possible, in practice it’s unlikely a terrorist organization would invest millions in an advanced genetic-engineering program. “I have been thinking quite a bit about bad things you could do with it, and we haven’t come up with anything that would succeed,” says Bier. “There are so many bad things you could do that are easier.”
Instead, Bier and James say they are convinced that engineered mosquitoes should be released as soon as possible, something they hope to do if they can find a community affected by malaria that will agree to it. “Imagine we could design a mosquito that would magically cure cancer,” says Bier. “Well, the fear of getting malaria is the same fear we have of getting cancer. In my opinion the benefits outweigh the risks, and we should move forward as aggressively as we can.”
It is distressing, but a fact, that the more rapidly any technology is adopted by scientists the more likely it is to leave people confused, anxious, and suspicious. This week, I wrote an article for the magazine about just such a revolutionary technique, called CRISPR, that permits scientists to edit the DNA of plants and animals with an ease and a precision that even a decade ago seemed inconceivable.
CRISPR research has already begun to transform molecular biology. There have been bold new claims about its promise and powers nearly every day. Yet, for the past fifty years, at least since Watson and Crick demonstrated that DNA contained the blueprints required to build everything alive, modern science has been caught in a hype trap. After all, if we possess such exquisitely detailed instructions, shouldn’t they be able to help us fix the broken genes that cause so many of our diseases?
The assumption has long been that the answer is yes. And for decades, we have been told (by the medical establishment, by pharmaceutical companies, and, sadly, by the press) that our knowledge of genetics will soon help us solve nearly every malady, whether it affects humans, other animals, or plants.
It turns out, however, that genetics and magic are two different things. Deciphering the blueprints in the three billion pairs of chemical letters which make up the human genome has been even more complex than anyone had imagined. And even though the advances have been real, and often dramatic, it doesn’t always seem that way. This has led many people to discount, and even fear, our most promising technologies. Somehow, we take lessons more readily from movies like “Jurassic Park” and “Gattaca” than from the very real, though largely incremental, advances in medical treatments.
This dangerous disconnect between scientific possibility and tangible results has already caused great harm: a scientifically unjustified fear of G.M.O.s, for example, has prevented many potentially life-enhancing crops from even being tested, let alone planted widely. The death of one patient, in 1999, halted all human-gene-therapy experiments in the United States for several years. We should, of course, be exceedingly cautious with such research, but if the U.S. is going to stop studies that could potentially help millions of people there are costs to that, too. (It’s worth remembering that there are real risks to everything we do: aspirin kills hundreds of Americans every year, and in the first half of 2015 nearly twenty thousand people have died in car accidents.)
Because it makes manipulating genes so much easier, CRISPR offers researchers the ability to rapidly accelerate studies of many types of illness, including cancers, autism, and AIDS. It will also make it possible to alter the genes of plants so that they can resist various diseases (without introducing the DNA of a foreign organism, which is how G.M.O.s are made). With CRISPR, almost anything could become possible: You want a unicorn? Just tweak the horse genome. How about a truly blue rose? The gene for the blue pigment does not exist naturally in roses. With CRISPR, it should be a trivial matter simply to edit that gene in.
Eventually, CRISPR should also permit technicians to edit embryos, which, at least in theory, could change the genetic lineage of mankind. The prospect is at least as frightening as it is exciting, and we need to start talking about that now. In the press, at least, that conversation—about perhaps the most exciting advance in the history of molecular biology—seems to have started. Two of the researchers I focussed on in my piece for The New Yorker have also been featured in other publications in the past two weeks: the Times has a profile of Jennifer Doudna, the Berkeley biochemist who helped figure out how to program CRISPR molecules to edit DNA, and STAT, a new online health and science publication launched by the Boston Globe’s owner, has one about Feng Zhang, a pioneering biologist at the Broad Institute of Harvard and M.I.T., who first made the technology work in mammals. The subject will soon get even more attention. Early next month, the National Academy of Sciences will convene an international conference devoted to the ethical use of this powerful new tool.
Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.
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Bringing Synthetic Biology to (Freeze-dried) Paper