The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. This review article discusses the potential of the modular design of novel regulatory proteins fashioned after the topology and mechanochemical properties of the lactose repressor.
The use of abstract chemical reaction networks (CRNs) as a modelling and design framework for the implementation of computing and control circuits using enzyme-free, entropy driven DNA strand displacement (DSD) reactions is starting to garner widespread attention in the area of synthetic biology. Previous work in this area has demonstrated the theoretical plausibility of using this approach to design biomolecular feedback control systems based on classical proportional-integral (PI) controllers, which may be constructed from CRNs implementing gain, summation and integrator operators. Here, we propose an alternative design approach that utilises the abstract chemical reactions involved in cellular signalling cycles to implement a biomolecular controller - termed a signalling-cycle (SC) controller. We compare the performance of the PI and SC controllers in closed-loop with a nonlinear second-order chemical process. Our results show that the SC controller outperforms the PI controller in terms of both performance and robustness, and also requires fewer abstract chemical reactions to implement, highlighting its potential usefulness in the construction of biomolecular control circuits.
"Since the description, in 2000, of two artificial gene networks, synthetic biology has emerged as a new engineering discipline that catalyzes a change of culture in the life sciences. Recombinant DNA can now be fabricated rather than cloned. Instead of focusing on the development of ad-hoc assembly strategies, molecular biologists can outsource the fabrication of synthetic DNA molecules to a network of DNA foundries. Model-driven product development cycles that clearly identify design, build, and test phases are becoming as common in the life sciences as they have been in other engineering fields. A movement of citizen scientists with roots in community labs throughout the world is trying to democratize genetic engineering. It challenges the life science establishment just like visionaries in the 70s advocated that computing should be personal at a time when access to computers was mostly the privilege of government scientists. Synthetic biology is a cultural revolution that will have far reaching implications for the biotechnology industry. The work of synthetic biologists today prefigures a new generation of cyber-biological systems that may to lead to the 5thindustrial revolution. By catering to the scientific publishing needs of all members of a diverse community, Synthetic Biology hopes to do its part to support the development of this new engineering discipline, catalyze the culture changes it calls for, and foster the development of a new industry far into the twenty first century."
Bioengineers have endowed a consortium of human cells with an artificial sense of smell, enabling the cells to detect, quantify, and remember the presence of gaseous volatile compounds in their environment.
DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.
Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering.
Date of Event 16th March 2017 Last Booking Date for this Event 9th March 2017 Places Available 100 Description 6:30pm - 9pm Thursday 16th March 2017
Bioengineering to produce complex control circuits like diagnostic tests, or to modify metabolic pathways for production of everything from drug and vaccines to flavours and fragrances, has typically taken place in cells that are then grown in large, industrial bioreactors. New methods, using cell extracts that can be programmed quickly and flexibly using DNA, promise a paradigm shift in biomanufacturing and pave the way to novel modes of computational biodesign, rapid prototyping and bioproduction. The opportunity to freeze-dry and ship these biofactories opens up many exciting possibilities for small scale distributed manufacturing, for example just-in-time vaccine production, and has profound implications for emerging bioeconomies.
The Synthetic Biology SRI welcomes two researchers to discuss this new area of synthetic biology and its possible futures.
Dr. Keith Pardee (University of Toronto) works at the interface of synthetic biology and human health. His research focuses on the potential of moving synthetic biology outside of the cell and dry shipment of programmable biofactories to enable diagnostics and just in time production of vaccines and biologics.
Dr. Richard Kelwick (Imperial College) researches cell-free systems and biopolymer production, including establishing cell-free methods and toolkits for new bacterial strains, most recently Bacillus subtilis. He also works on bioreporters and biosensors using synthetic gene circuits.
The talk and dialogue will be followed by a wine reception and delicious finger buffet.
This week the US Patent Trial and Appeal Board decided that there is “no interference in fact” between the patent application of Jennifer Doudna and University of California and the patents granted to Feng Zhang and the Broad Institute of Harvard and MIT. You can read the full text of the decision here.
Media reaction: Hype!
The immediate reactions put this as a big win for the Broad Institute in as many combative terms as possible. Fight, battle, and war were all used to describe the legal patent interference case being settled and most headlines pronounced the war to be over. Obviously this has been a huge court battle worth enough to merit legal bills going into the tens of millions of dollars, but many experts have noted this is not necessarily the winner-take-all patent fight that many headlines suggest. The interference proceedings were to determine if the set of patents granted to the Broad Institute for CRISPR-Cas9 editing in eukaryotic cells interfere with the patent application from the University of California (UC) that was filed first and tries to generally cover the use of CRISPR-Cas9 in living cells.
Word cloud of 15 top CRISPR patent headlines made using http://wordcloud.cs.arizona.edu/index.html This decision does mean that barring a successful appeal at the Federal Circuit Appeals Court, the Broad Institute will get to keep the patents using Cas9 to do genome editing in eukaryotic cells–which includes human cells–so that is key to companies that had licensed those patents to develop human therapeutics. The UC patent application tries to make a broader claim to editing in all living cells, which would naturally include eukaryotic and human cells. If UC gets the patent on the broad use in living cells and the Broad has the patents on use in eukaryotic cells, we may still end up in a scenario in which licensing of both patents is necessary to edit eukaryotic cells. Some good reporting that helped me understand this can be found here, here, here, here, and here. After the initial rush to announce the epic end to this patent battle, time allowed for more nuanced and thorough reporting to lay out the remaining uncertainty and caveats.
Market reaction: Follow the hype!
Following the main narrative of the Broad Institute scoring a big win in its ‘epic battle’, the market traders pushed Editas (aligned with Broad) stock about 30% throughout the day and Intellia (aligned with UC) stock dropped nearly 10%. It’s hard for me to tell how much the decision actually has on the value of these companies because it seems like that will depend on how licensing or cross-licensing options work out for any given application. I’ve yet to find much analysis that could definitively map out how the patent licensing will affect medical innovation using CRISPR-Cas9, but that may still be too variable for anyone to know right now.
There was a lot of discussions of the possible ways this could play out in the biotech and gene editing industries depending on the course that UC takes after the interference defeat. The tweet below from CNBC reporter Meg Tirrell outlines three possible options.
Living systems, such as bacteria, yeasts, and mammalian cells, can be genetically programmed with synthetic circuits that execute sensing, computing, memory, and response functions. Integrating these functional living components into materials and devices will provide powerful tools for scientific research and enable new technological applications. However, it has been a grand challenge to maintain the viability, functionality, and safety of living components in freestanding materials and devices, which frequently undergo deformations during applications. Here, we report the design of a set of living materials and devices based on stretchable, robust, and biocompatible hydrogel–elastomer hybrids that host various types of genetically engineered bacterial cells. The hydrogel provides sustainable supplies of water and nutrients, and the elastomer is air-permeable, maintaining long-term viability and functionality of the encapsulated cells. Communication between different bacterial strains and with the environment is achieved via diffusion of molecules in the hydrogel. The high stretchability and robustness of the hydrogel–elastomer hybrids prevent leakage of cells from the living materials and devices, even under large deformations. We show functions and applications of stretchable living sensors that are responsive to multiple chemicals in a variety of form factors, including skin patches and gloves-based sensors. We further develop a quantitative model that couples transportation of signaling molecules and cellular response to aid the design of future living materials and devices.
Several institutions are embroiled in a legal dispute over the foundational patent rights to CRISPR-Cas9 gene-editing technology, and it may take years for their competing claims to be resolved (1–4). But even before ownership of the patents is finalized, the institutions behind CRISPR have wasted no time capitalizing on the huge market for this groundbreaking technology by entering into a series of license agreements with commercial enterprises (see the figure). With respect to the potentially lucrative market for human therapeutics and treatments, each of the key CRISPR patent holders has granted exclusive rights to a spinoff or “surrogate” company formed by the institution and one of its principal researchers (5, 6). Although this model, in which a university effectively outsources the licensing and commercialization of a valuable patent portfolio to a private company, is not uncommon in the world of university technology transfer, we suggest it could rapidly bottleneck the use of CRISPR technology to discover and develop useful human therapeutics.
The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein–ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure–function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI.
To find experimental validation for electrostatic interactions essential for catalytic reactions represents a challenge due to practical limitations in assessing electric fields within protein structures. SCOPE OF REVIEW: This review examines the applications of non-canonical amino acids (ncAAs) as genetically encoded probes for studying the role of electrostatic interactions in enzyme catalysis. MAJOR CONCLUSIONS: ncAAs constitute sensitive spectroscopic probes to detect local electric fields by exploiting the vibrational Stark effect (VSE) and thus have the potential to map the protein electrostatics. GENERAL SIGNIFICANCE: Mapping the electrostatics in proteins will improve our understanding of natural catalytic processes and, in beyond, will be helpful for biocatalyst engineering. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation.
By: Emmette Hutchinson, PhD Synthetic biology is an interdisciplinary field that utilizes an engineering approach to construct novel biological products, circuits and designer organisms. This field has the potential to revolutionize many aspects of society from chemical production to healthcare. Synthetic biology holds particular promise in the production of biological therapeutics or chemical compounds…
Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5 g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40 g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1 g/L at 24 h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62 g/L/h (which was 0.22 g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.
Extensive evidence has shown that long-range charge transport can occur along double helical DNA, but active control (switching) of single-DNA conductance with an external field has not yet been demonstrated. Here we demonstrate conductance switching in DNA by replacing a DNA base with a redox group. By applying an electrochemical (EC) gate voltage to the molecule, we switch the redox group between the oxidized and reduced states, leading to reversible switching of the DNA conductance between two discrete levels. We further show that monitoring the individual conductance switching allows the study of redox reaction kinetics and thermodynamics at single molecular level using DNA as a probe. Our theoretical calculations suggest that the switch is due to the change in the energy level alignment of the redox states relative to the Fermi level of the electrodes.
Genetic information in genomes is ordered, arranged in such a way that it constitutes a code, the so-called cis regulatory code. The regulatory machinery of the cell, termed trans-factors, decodes and expresses this information. In this way, genomes maintain a potential repertoire of genetic programs, parts of which are executed depending on the presence of active regulators in each condition. These genetic programs, executed by the regulatory machinery, have functional units in the genome delimited by punctuation-like marks. In genetic terms, these informational phrases correspond to transcription units, which are the minimal genetic information expressed consistently from initiation to termination marks. Between the start and final punctuation marks, additional marks are present that are read by the transcriptional and translational machineries. In this work, we look at all the experimentally described and predicted genetic elements in the bacterium Escherichia coli K-12 MG1655 and define a comprehensive architectural organization of transcription units to reveal the natural genome-design and to guide the construction of synthetic genetic programs.
If you are a scientist, in particular a biologist, you might be very familiar with the frustration that comes from trying to reproduce an experiment described in a paper authored by someone else. No matter the journal where the research is published, reproducibility in science is one of the practitioner’s biggest concerns. A recent survey carried out by Nature involving 1576 researchers states that ‘more than 70% of researchers [involved in the survey] have tried and failed to reproduce another scientist’s experiments, and more than half have failed to reproduce their own experiments.’
But does this mean that most of the published research is wrong or untrustworthy? Not necessarily. The same study suggests two reasons behind these reproducibility issues:
the complexity of science: sometimes slight modification in the conditions in which the experiment is carried out might make a huge difference Selective reporting: cherry picking data in support of a hypothesis, for instance. When asked to select from a list of potential solutions, 90% of the Nature survey respondents choose better mentorship, proper use of statistics, and more robust experimental design.
Synthace Ltd., a UK based technology company is already working on the development of a platform that could help ‘biology engineers’ solve the problem by providing the means to interact with lab equipment and automation in a much more flexible and high level way than is currently possible. ‘Automation is key because of both its intrinsic repeatability and the experimental bandwidth it provides to do better designed experiments,’ says Tim (CEO of Synthace).
When Synthace was founded in 2011, it began life as a synthetic biology company looking to exploit the huge power of biology to manufacture novel products. However, they soon realized that they did not have the necessary tools to do this. They, therefore, proceeded to invent those tools, and in the process they came to understand that the platform they developed could be invaluable for the entire community. Synthace is now a software company for biology, focused on the development of a transformative technology that will allow whoever wants to engineer biology to do so in a much more reliable way and exploiting the power of biological complexity more thoroughly (for instance, considering many more experimental variables at the same time).
STEP INTO ASSOCIATE PROFESSOR MARK BATHE’S LAB in the Department of Biological Engineering and you’ll find design and biology merging at the nanoscale. Under an electron microscope you might see a cornucopia of three-dimensional shapes—icosahedra, pyramids, and stars—all assembled from synthetic strands of DNA. “There’s no other molecular medium we can design and fabricate with such a versatility of geometries and precision at the nanoscale as DNA,” says Bathe ’98, SM ’01, PhD ’04.
LEARN MORE DAEDALUS And Bathe has added to that versatility: with graduate student Sakul Ratanalert, he recently developed software called DAEDALUS, which captures the complex rules of DNA construction in an algorithm that makes three-dimensional DNA design easier and more accessible to a wide range of scientists and engineers.
Most people think of the spiraling set of nucleic acids purely as the code of life. The strings of As, Ts, Cs, and Gs (adenine, thymine, cytosine, and guanine) in cells provide the blueprint for how living things behave and reproduce. And for nearly half a century bioengineers have creatively manipulated those sequences to change the way organisms function—breeding new pest-resistant plants, for example, or microbes that ferment medicines and chemicals.
But the double helix of DNA also possesses unique characteristics as a nano-building material. In 2006, Caltech researcher Paul Rothemund discovered that if he synthesized DNA letters in specific sequences, the molecular bonds that glue the As to Ts and Cs to Gs, and which come undone when DNA replicates, could be used to fold the DNA into two- and three-dimensional shapes. With a nod to both the precision and elegance of the technique, scientists dubbed it “DNA origami.”
The beauty of DNA origami is that once the components are collected, all it takes is a little shake and some Brownian motion—the random movement of particles in fluid—for these shapes to assemble themselves. The system uses a single long strand of DNA as scaffolding on which to stick smaller strings of letters. The DNA conforms to the shape as the letters bond to each other.
The beauty of DNA origami is that once the components are collected, all it takes is a little shake and some Brownian motion—the random movement of particles in fluid—for these shapes to assemble themselves. But the rules for designing DNA origami are difficult, if not arcane, and lining up nucleotides to fold into corresponding 3-D shapes can tax even the most brilliant minds. “It’s been limited to a small group of experts,” Bathe says. His software is changing that.
Rather than manually fiddling with sequences of nucleotides, DAEDALUS users design the target geometric structures they want, and the algorithm generates the corresponding nucleotide sequences to make them. “You give the software a high-level geometric shape, and then it will automatically produce that shape using DNA,” Bathe says.
In a sense, Bathe himself may be a perfect researcher for exploring how these geometries translate across scales. He’s part of an MIT legacy—the son of longtime engineering faculty member Klaus-Jürgen Bathe. Like his father, the younger Bathe earned his PhD in mechanical engineering; but from childhood, his interests skewed towards biology and medicine. “I’ve always wanted to build technologies that impact human health, more than cars or bridges, like my father,” he says. With DAEDALUS, Bathe has built a bridge of another kind, connecting designers of many disciplines with the tools of molecular biology.
Bathe is now working to harness his DNA nanoshapes to deliver drugs inside the body. Taking a cue from viruses that attach to cells to infect them, Bathe hopes to design a variety of DNA structures that deliver payloads of antibodies or even gene-editing enzymes such as Cas9 to diseased cells within the body. “The holy grail would be to edit the brain for treatment of diseases such as autism or schizophrenia, or cancer cells in malignant tumors,” Bathe says.
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.
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