It was Wednesday 16 February 2011, and Goldman was at a hotel in Hamburg, Germany, talking with some of his fellow bioinformaticists about how they could afford to store the reams of genome sequences and other data the world was throwing at them. He remembers the scientists getting so frustrated by the expense and limitations of conventional computing technology that they started kidding about sci-fi alternatives. “We thought, 'What's to stop us using DNA to store information?'”
Then the laughter stopped. “It was a lightbulb moment,” says Goldman, a group leader at the European Bioinformatics Institute (EBI) in Hinxton, UK. True, DNA storage would be pathetically slow compared with the microsecond timescales for reading or writing bits in a silicon memory chip. It would take hours to encode data by synthesizing DNA strings with a specific pattern of bases, and still more hours to recover that information using a sequencing machine. But with DNA, a whole human genome fits into a cell that is invisible to the naked eye. For sheer density of information storage, DNA could be orders of magnitude beyond silicon — perfect for long-term archiving.
“We sat down in the bar with napkins and biros,” says Goldman, and started scribbling ideas: “What would you have to do to make that work?” The researchers' biggest worry was that DNA synthesis and sequencing made mistakes as often as 1 in every 100 nucleotides. This would render large-scale data storage hopelessly unreliable — unless they could find a workable error-correction scheme. Could they encode bits into base pairs in a way that would allow them to detect and undo the mistakes? “Within the course of an evening,” says Goldman, “we knew that you could.”
He and his EBI colleague Ewan Birney took the idea back to their labs, and two years later announced that they had successfully used DNA to encode five files, including Shakespeare's sonnets and a snippet of Martin Luther King's 'I have a dream' speech1. By then, biologist George Church and his team at Harvard University in Cambridge, Massachusetts, had unveiled an independent demonstration of DNA encoding2. But at 739 kilobases (kB), the EBI files comprised the largest DNA archive ever produced — until July 2016, when researchers from Microsoft and the University of Washington claimed a leap to 200 megabytes (MB).
The latest experiment signals that interest in using DNA as a storage medium is surging far beyond genomics: the whole world is facing a data crunch. Counting everything from astronomical images and journal articles to YouTube videos, the global digital archive will hit an estimated 44 trillion gigabytes (GB) by 2020, a tenfold increase over 2013. By 2040, if everything were stored for instant access in, say, the flash memory chips used in memory sticks, the archive would consume 10–100 times the expected supply of microchip-grade silicon3.
That is one reason why permanent archives of rarely accessed data currently rely on old-fashioned magnetic tapes. This medium packs in information much more densely than silicon can, but is much slower to read. Yet even that approach is becoming unsustainable, says David Markowitz, a computational neuroscientist at the US Intelligence Advanced Research Projects Activity (IARPA) in Washington DC. It is possible to imagine a data centre holding an exabyte (one billion gigabytes) on tape drives, he says. But such a centre would require US$1 billion over 10 years to build and maintain, as well as hundreds of megawatts of power. “Molecular data storage has the potential to reduce all of those requirements by up to three orders of magnitude,” says Markowitz. If information could be packaged as densely as it is in the genes of the bacterium Escherichia coli, the world's storage needs could be met by about a kilogram of DNA (see 'Storage limits').
Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications.
We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable.
The molecular components of transcriptional regulation are modular. Transcription factors have domains for specific functions such as DNA binding, dimerization, and protein-protein interactions associated with transcriptional activation and repression. Similarly, promoters are modular. They consist of combinations of cis-acting elements that are the binding sites for transcription factors. It is this promoter architecture that largely determines the expression pattern of a gene. The modular nature of promoters is supported by the observation that many cis-acting elements retain their activities when they are taken out of their native promoter context and used as building blocks in synthetic promoters. We therefore have a large collection of cis-acting elements to use in building synthetic promoters and many minimal promoters upon which to build them. This review discusses what we have learned concerning how to use these building blocks to make synthetic promoters. It has become clear that we can increase the strength of a promoter by adding increasing numbers of cis-acting elements. However, it appears that there may be a sweet spot with regard to inducibility as promoters with increasing numbers of copies of an element often show increased background expression. Spacing between elements appears important because if elements are placed too close together activity is lost, presumably due to reduced transcription factor binding due to steric hindrance. In many cases, promoters that contain combinations of cis-acting elements show better expression characteristics than promoters that contain a single type of element. This may be because multiple transcription factor binding sites in the promoter places it at the end of multiple signal transduction pathways. Finally, some cis-acting elements form functional units with other elements and are inactive on their own. In such cases, the complete unit is required for function in a synthetic promoter. Taken together, we have learned much about how to construct synthetic promoters and this knowledge will be crucial in both designing promoters to drive transgenes and also as components of defined regulatory networks in synthetic biology.
Biology is the world’s greatest manufacturing platform, according to MIT spinout Ginkgo Bioworks. The synthetic-biology startup is re-engineering yeast to act as tiny organic “factories” that produce chemicals for the flavor, fragrance, and food industries, with aims of making products more quickly, cheaply, and efficiently than traditional methods. “We see biology as a transformative technology,” says Ginkgo co-founder Reshma Shetty PhD ’08, who co-invented the technology at MIT. “It is the most powerful and sophisticated manufacturing platform on the planet, able to self-assemble incredible structures at a scale that is far out of reach of the most cutting-edge human technology.” Similar to how beer is brewed — where yeast eats sugars and creates alcohol and flavors through fermentation — Ginkgo’s “hacked” yeast eats fatty acids and produces desired chemicals that recreate certain scents and flavors during fermentation. Those chemicals can then be extracted and used in a number of different products.
The genome-editing system known as CRISPR allows scientists to delete or replace any target gene in a living cell. MIT researchers have now added an extra layer of control over when and where this gene editing occurs, by making the system responsive to light. With the new system, gene editing takes place only when researchers shine ultraviolet light on the target cells. This kind of control could help scientists study in greater detail the timing of cellular and genetic events that influence embryonic development or disease progression. Eventually, it could also offer a more targeted way to turn off cancer-causing genes in tumor cells. “The advantage of adding switches of any kind is to give precise control over activation in space or time,” says Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Electrical Engineering and Computer Science at MIT and a member of MIT’s Koch Institute for Integrative Cancer Research and its Institute for Medical Engineering and Science. Bhatia is the senior author of a paper describing the new technique in the journal Angewandte Chemie. The paper’s lead author is Piyush Jain, a postdoc in MIT’s Institute for Medical Engineering and Science. Light sensitivity Before coming to MIT, Jain developed a way to use light to control a process called RNA interference, in which small strands of RNA are delivered to cells to temporarily block specific genes. “While he was here, CRISPR burst onto the scene and he got very excited about the prospect of using light to activate CRISPR in the same way,” Bhatia says. CRISPR relies on a gene-editing complex composed of a DNA-cutting enzyme called Cas9 and a short RNA strand that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. When Cas9 and the guide RNA are delivered into cells, a specific cut is made in the genome; the cells’ DNA repair processes glue the cut back together but permanently delete a small portion of the gene, making it inoperable. In previous efforts to create light-sensitive CRISPR systems, researchers have altered the Cas9 enzyme so that it only begins cutting when exposed to certain wavelengths of light. The MIT team decided to take a different approach and make the binding of the RNA guide strand light-sensitive. For possible future applications in humans, it could be easier to deliver these modified RNA guide strands than to program the target cells to produce light-sensitive Cas9, Bhatia says. “You really don’t have to do anything different with the cargo you were planning to deliver except to add the light-activated protector,” she says. “It’s an attempt to make the system much more modular.” To make the RNA guide strands light-sensitive, the MIT team created “protectors” consisting of DNA sequences with light-cleavable bonds along their backbones. These DNA strands can be tailored to bind to different RNA guide sequences, forming a complex that prevents the guide strand from attaching to its target in the genome. When the researchers expose the target cells to light with a wavelength of 365 nanometers (in the ultraviolet range), the protector DNA breaks into several smaller segments and falls off the RNA, allowing the RNA to bind to its target gene and recruit Cas9 to cut it. Targeting multiple genes In this study, the researchers demonstrated that they could use light to control editing of the gene for green fluorescent protein (GFP) and two genes for proteins normally found on cell surfaces and overexpressed in some cancers. “If this is really a generalizable scheme, then you should be able to design protector sequences against different target sequences,” Bhatia says. “We designed protectors against different genes and showed that they all could be light-activated in this way. And in a multiplexed experiment, when a mixed population of protectors was used, the only targets that were cleaved after light exposure were those being photo-protected.” This precise control over the timing of gene editing could help researchers study the timing of cellular events involved in disease progression, in hopes of determining the best time to intervene by turning off a gene. “CRISPR-Cas9 is a powerful technology that scientists can use to study how genes affect cell behavior,” says James Dahlman, an assistant professor of biomedical engineering at Georgia Tech, who was not involved in the research. “This important advance will enable precise control over those genetic changes. As a result, this work gives the scientific community a very useful tool to advance many gene editing studies.” Bhatia’s lab is also pursuing medical applications for this technique. One possibility is using it to turn off cancerous genes involved in skin cancer, which is a good target for this approach because the skin can be easily exposed to ultraviolet light. The team is also working on a “universal protector” that could be used with any RNA guide strand, eliminating the need to design a new one for each RNA sequence, and allowing it to inhibit CRISPR-Cas9 cleavage of many targets at once. The research was funded by the Ludwig Center for Molecular Oncology, the Marie-D. and Pierre Casimir-Lambert Fund, a Koch Institute Support Grant from the National Cancer Institute, and the Marble Center for Cancer Nanomedicine.
The dielectric response of a material is central to numerous processes spanning the fields of chemistry, materials science, biology, and physics. Despite this broad importance across these disciplines, describing the dielectric environment of a molecular system at the level of first-principles theory and computation remains a great challenge and is of importance to understand the behavior of existing systems as well as to guide the design and synthetic realization of new ones. Furthermore, with recent advances in molecular electronics, nanotechnology, and molecular biology, it has become necessary to predict the dielectric properties of molecular systems that are often difficult or impossible to measure experimentally. In these scenarios, it is would be highly desirable to be able to determine dielectric response through efficient, accurate, and chemically informative calculations.
A good example of where theoretical modeling of dielectric response would be valuable is in the development of high-capacitance organic gate dielectrics for unconventional electronics such as those that could be fabricated by high-throughput printing techniques. Gate dielectrics are fundamental components of all transistor-based logic circuitry, and the combination high dielectric constant and nanoscopic thickness (i.e., high capacitance) is essential to achieving high switching speeds and low power consumption. Molecule-based dielectrics offer the promise of cheap, flexible, and mass producible electronics when used in conjunction with unconventional organic or inorganic semiconducting materials to fabricate organic field effect transistors (OFETs). The molecular dielectrics developed to date typically have limited dielectric response, which results in low capacitances, translating into poor performance of the resulting OFETs. Furthermore, the development of better performing dielectric materials has been hindered by the current highly empirical and labor-intensive pace of synthetic progress. An accurate and efficient theoretical computational approach could drastically decrease this time by screening potential dielectric materials and providing reliable design rules for future molecular dielectrics.
Until recently, accurate calculation of dielectric responses in molecular materials was difficult and highly approximate. Most previous modeling efforts relied on classical formalisms to relate molecular polarizability to macroscopic dielectric properties. These efforts often vastly overestimated polarizability in the subject materials and ignored crucial material properties that can affect dielectric response. Recent advances in first-principles calculations via density functional theory (DFT) with periodic boundary conditions have allowed accurate computation of dielectric properties in molecular materials.
In this Account, we outline the methodology used to calculate dielectric properties of molecular materials. We demonstrate the validity of this approach on model systems, capturing the frequency dependence of the dielectric response and achieving quantitative accuracy compared with experiment. This method is then used as a guide to new high-capacitance molecular dielectrics by determining what materials and chemical properties are important in maximizing dielectric response in self-assembled monolayers (SAMs). It will be seen that this technique is a powerful tool for understanding and designing new molecular dielectric systems, the properties of which are fundamental to many scientific areas.
"Human exploration off planet is severely limited by the cost of launching materials into space and by re-supply. Thus materials brought from Earth must be light, stable and reliable at destination. Using traditional approaches, a lunar or Mars base would require either transporting a hefty store of metals or heavy manufacturing equipment and construction materials for in situ extraction; both would severely limit any other mission objectives. Long-term human space presence requires periodic replenishment, adding a massive cost overhead. Even robotic missions often sacrifice science goals for heavy radiation and thermal protection. Biology has the potential to solve these problems because life can replicate and repair itself, and perform a wide variety of chemical reactions including making food, fuel and materials. Synthetic biology enhances and expands life's evolved repertoire. Using organisms as feedstock, additive manufacturing through bioprinting will make possible the dream of producing bespoke tools, food, smart fabrics and even replacement organs on demand. This new approach and the resulting novel products will enable human exploration and settlement on Mars, while providing new manufacturing approaches for life on Earth."
Advances in synthetic biology have enabled the engineering of cells with genetic circuits in order to program cells with new biological behavior, dynamic gene expression, and logic control. This cellular engineering progression offers an array of living sensors that can discriminate between cell states, produce a regulated dose of therapeutic biomolecules, and function in various delivery platforms. In this review, we highlight and summarize the tools and applications in bacterial and mammalian synthetic biology. The examples detailed in this review provide insight to further understand genetic circuits, how they are used to program cells with novel functions, and current methods to reliably interface this technology in vivo; thus paving the way for the design of promising novel therapeutic applications.
Biological and synthetic recognition elements are at the heart of the majority of modern bioreceptor assays. Traditionally, enzymes and antibodies have been integrated in the biosensor designs as a popular choice for the detection of toxin molecules. But since 1970s, alternative biological and synthetic binders have been emerged as a promising alternative to conventional biorecognition elements in detection systems for laboratory and field-based applications. Recent research has witnessed immense interest in the use of recombinant enzymatic methodologies and nanozymes to circumvent the drawbacks associated with natural enzymes. In the area of antibody production, technologies based on the modification of in vivo synthesized materials and in vitro approaches with development of “display “systems have been introduced in the recent years. Subsequently, molecularly-imprinted polymers and Peptide nucleic acid (PNAs) were developed as an attractive receptor with applications in the area of sample preparation and detection systems. In this article, we discuss all alternatives to conventional biomolecules employed in the detection of various toxin molecules We review recent developments in modified enzymes, nanozymes, nanobodies, aptamers, peptides, protein scaffolds and DNazymes. With the advent of nanostructures and new interface materials, these recognition elements will be major players in future biosensor development.
Steven Burgess steps down as managing editor at the PLOS SynBio Community. His parting review is typically excellent. Great piece on Oxitec’s sterile mosquitos, and the human challenge of testing them in Key West. NIH reconsiders its moratorium on human-animal chimera research. A PBS article on using CRISPR in grapes turns into a remarkably comprehensive overview of the opportunities and challenges in editing agricultural plants. Is NgAgo’s gene editing reproducible? Jury’s still out, but preliminary evidence isn’t good. DARPA launches an Engineered Living Materials initiative, with the goal of designing biomaterials that grow into the shape of whatever structure/component. Open for proposals through September! Interesting warning about over-hyping technology: a piece on embryonic stem cells and the challenges of treating disease with them. If you’re in New York and want to build stuff with bio, you can now take a crash course in making custom biomaterials, put on by New York’s Genspace DIYbio lab. If you find yourself in Cambridge, check out Cafe Synthetique, the local synbio salon. Sounds like a GeneMods sister organization! Cool profile in STAT of David Baker and co.’s plans to solve problems in the world (mostly diseases, in this piece) with computationally designed proteins. Highlights from the 2016 BioDesign Challenge (from last month, but good enough to post anyway). Industry news:
Engineered Cas9 with lower off-target effects goes commercial: high-fidelity Cas9 variant from Joung lab licensed to Editas. Total cost of CRISPR patent fight exceeds $20 million and counting. DOD gives Ginkgo $2 million to develop probiotic vaccine for traveler’s diarrhea. Books and Longreads
I just discovered BioCoder, a quarterly newsletter about synthetic biology from technology media company O’Reilly Media, and I’m really enjoying it. Carl Zimmer gets his genome sequenced and analyzed by some of the best researchers in the field. A Game of Genomes chronicles his adventure. A must-read. Ed Yong’s fantastic book about the microbes that precede, surround, live on, and comprise us, I Contain Multitudes, came out. I cannot recommend enough that you get your hands (or ears, it’s on Audible!) on this book. Now, onto the research papers!
Easily and quickly sensing the presence of small molecules is one of the rate-limiting challenges in metabolic engineering. Now, Savage Lab reports rapid construction of metabolite biosensors using domain insertion profiling. Baker Lab does it again, reporting rationally designed, two-component, 120 subunit, icosahedral protein complexes.. Electrochemical across membranes fundamentally underly pretty much all biological energy production. Fotiadis Lab has now engineered a photo-switchable, light-driven proton pump. Genetic circuits:
A Lu Lab collaboration programs yeast and builds a mini bioreactor to produce single doses of multiple biological therapeutics on command. CRISPR/Gene Editing:
A Joung Lab collaboration relaxes Staphylococcus aureus Cas9’s PAM specificity, expanding the sites this smaller programmable nuclease can target. It’s been a bumper crop month for making molecular recordings in the genomes of cells. Science published three papers (summarized here): Church Lab reported molecular recordings produced using the actual CRISPR parts of CRISPR (as opposed to Cas9); Lu Lab used recombinases to build state machines in living cells; and a collaboration between Shendure and Schier Labs used Cas9 to record the differentiation and trace the lineage of all the cells in a zebrafish. Then, Lu Lab developed a method to continuously record the presence of Cas9 in human cells using a self-targeting guide RNA. Metabolic Engineering
Jewett Lab takes a step toward easier natural product mining by expressing non-ribosomal peptide synthetases and producing small-molecule peptides in a cell-free lysate system. Building biology to understand it
A massively recoded, 57-codon E. coli? Not quite yet, but Church Lab computationally designs the genome, synthesizes the parts, and makes progress on assembling and testing them. Venter Institute edits and interrogates bacterial ribosome genes, on a synthetic bacterial genome in yeast. GenomeWeb has a summary if you don’t have time to read the whole paper. Review article in ACS SynBio argues that mammalian artificial chromosomes (MACs) are the way of the future.
Powered by a chemical reaction controlled by microfluidics, the 3D-printed 'octobot' has no electronics
By Leah Burrows, SEAS Communications
(CAMBRIDGE, Massachusetts) — A team of Harvard University researchers with expertise in 3D printing, mechanical engineering, and microfluidics has demonstrated the first autonomous, untethered, entirely soft robot. This small, 3D-printed robot — nicknamed the octobot — could pave the way for a new generation of completely soft, autonomous machines.
Eventbrite - ThinkSTEAM presents Synthetic Biology Workshop with Columbia University's iGEM Team - Saturday, October 1, 2016 at Lasker Biomedical Research Building, New York, NY. Find event and ticket information.
Algae (a term used to group many photosynthetic organisms into a rather heterologous mash-up) do not have a kind place in the public imagination. Take for example the following passage from Stephen King’s Pet Semetary:
“Dead fields under a November sky, scattered rose petals brown and turning up at the edges, empty pools scummed with algae, rot, decomposition, dust… “
Leaving aside their use in a horror novel as a way to set an image of decay, algae attract significant scientific attention, and are even considered cool (well, at least by some). But when I tell people I work on algal Synbio, one question that eventually comes up (or is silently implied) is why do I bother, when there are more prominent and better-developed systems? In this blog I will try to partly address this point of view, as well as provide a small perspective on the subject.
The practical benefits of algae for humans date back to the days of early photosynthesis, when they produced the oxygenic atmosphere we currently breathe. Moreover, algal forms were the ancestors of chloroplasts and land plants. Today, algae are found in almost every environment, in having various forms and ecological roles. But what is their place in synthetic biology?
In terms of algal synthetic biology and biotechnology, there are two main research domains: eukaryotic microalgae and cyanobacteria.
The model for studying eukaryotic microalgae is Chlamydomonas reinhardtii—although the use of more species is being explored. C. reinhardtii can be transformed both via nuclear and chloroplast transformation, each having different potentials (e.g. random insertion vs. homologous recombination, eukaryotic vs. prokaryotic translation and protein maturation, etc.)
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