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Secrets of the CRISPR Arms Race

Secrets of the CRISPR Arms Race | SynBioFromLeukipposInstitute | Scoop.it
Anti-CRISPR proteins analyzed by cryo-electron microscopy reveal their structural and functional secrets
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Can a new model help governance keep up with synthetic biology? | PLOS Synthetic Biology Community

Can a new model help governance keep up with synthetic biology? | PLOS Synthetic Biology Community | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is moving at a rapid pace, and regulations will need to keep up to allow useful technologies to hit the market while maintaining a high standard of safety. Since most policymakers are not experts in all of the newest synthetic biology technologies, better analysis tools are needed to understand how to react. So two researchers, Christopher Cummings and Jennifer Kuzma, from North Carolina State University and Nanyang Technological University built a model to determine how to prepare for handling the regulation new synthetic biology products.

Building a model for governing new synbio technology

This model for assessing risks of new synthetic biology technologies is called Societal Risk Evaluation Scheme (SRES) and it tries to make governance more anticipatory than just reactive. This is a tough problem. How do we predict the risks of technologies that don’t exist yet? How do we assess products that seem totally new? The field of synthetic biology is pushing the boundaries of what we can do with and to biology.
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Diversity, Application, and Synthetic Biology of Industrially Important Aspergillus Fungi

The filamentous fungal genus Aspergillus consists of over 340 officially recognized species. A handful of these Aspergillus fungi are predominantly used for food fermentation and large-scale production of enzymes, organic acids, and bioactive compounds. These industrially important Aspergilli primarily belong to the two major Aspergillus sections, Nigri and Flavi. Aspergillus oryzae (section Flavi) is the most commonly used mold for the fermentation of soybeans, rice, grains, and potatoes. Aspergillus niger (section Nigri) is used in the industrial production of various enzymes and organic acids, including 99% (1.4 million tons per year) of citric acid produced worldwide. Better understanding of the genomes and the signaling mechanisms of key Aspergillus species can help identify novel approaches to enhance these commercially significant strains. This review summarizes the diversity, current applications, key products, and synthetic biology of Aspergillus fungi commonly used in industry.
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UC Berkeley Receives CRISPR Patent in Europe | The Scientist Magazine®

UC Berkeley Receives CRISPR Patent in Europe | The Scientist Magazine® | SynBioFromLeukipposInstitute | Scoop.it
The European Patent Office will grant patent rights over the use of CRISPR in all cell types to a University of California team, contrasting with a recent decision in the U.S.
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Efforts and Challenges in Engineering the Genetic Code

This year marks the 48th anniversary of Francis Crick's seminal work on the origin of the genetic code, in which he first proposed the "frozen accident" hypothesis to describe evolutionary selection against changes to the genetic code that cause devastating global proteome modification. However, numerous efforts have demonstrated the viability of both natural and artificial genetic code variations. Recent advances in genetic engineering allow the creation of synthetic organisms that incorporate noncanonical, or even unnatural, amino acids into the proteome. Currently, successful genetic code engineering is mainly achieved by creating orthogonal aminoacyl-tRNA/synthetase pairs to repurpose stop and rare codons or to induce quadruplet codons. In this review, we summarize the current progress in genetic code engineering and discuss the challenges, current understanding, and future perspectives regarding genetic code modification.
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De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds

De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds | SynBioFromLeukipposInstitute | Scoop.it
The Zika outbreak, spread by the Aedes aegypti mosquito, highlights the need to create high-quality assemblies of large genomes in a rapid and cost-effective fashion. Here, we combine Hi-C data with existing draft assemblies to generate chromosome-length scaffolds. We validate this method by assembling a human genome, de novo, from short reads alone (67X coverage). We then combine our method with draft sequences to create genome assemblies of the mosquito disease vectors Aedes aegypti and Culex quinquefasciatus, each consisting of three scaffolds corresponding to the three chromosomes in each species. These assemblies indicate that virtually all genomic rearrangements among these species occur within, rather than between, chromosome arms. The genome assembly procedure we describe is fast, inexpensive, accurate, and can be applied to many species.
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Towards single molecule biosensors using super-resolution fluorescence microscopy

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.
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Smartphone-based sensing system using ZnO and graphene modified electrodes for VOCs detection

Volatile organic compounds (VOCs) detection is in high demand for clinic treatment, environment monitoring, and food quality control. Especially, VOCs from human exhaled breath can serve as significant biomarkers of some diseases, such as lung cancer and diabetes. In this study, a smartphone-based sensing system was developed for real-time VOCs monitoring using alternative current (AC) impedance measurement. The interdigital electrodes modified with zinc oxide (ZnO), graphene, and nitrocellulose were used as sensors to produce impedance responses to VOCs. The responses could be detected by a hand-held device, sent out to a smartphone by Bluetooth, and reported with concentration on an android program of the smartphone. The smartphone-based system was demonstrated to detect acetone at concentrations as low as 1.56 ppm, while AC impedance spectroscopy was used to distinguish acetone from other VOCs. Finally, measurements of the exhalations from human being were carried out to obtain the concentration of acetone in exhaled breath before and after exercise. The results proved that the smartphone-based system could be applied on the detection of VOCs in real settings for healthcare diagnosis. Thus, the smartphone-based system for VOCs detection provided a convenient, portable and efficient approach to monitor VOCs in exhaled breath and possibly allowed for early diagnosis of some diseases.
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Patent pools for CRISPR technology

Patent pools for CRISPR technology | SynBioFromLeukipposInstitute | Scoop.it
J. L. Contreras and J. S. Sherkow's Policy Forum “CRISPR, surrogate licensing, and scientific discovery” (17 February, p. [698][1]) suggests that exclusive licenses granted by the foundational patent holders “could rapidly bottleneck the use of CRISPR technology to discover and develop useful
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Biohackers Are Engineering Yeast to Make THC

Biohackers Are Engineering Yeast to Make THC | SynBioFromLeukipposInstitute | Scoop.it
The THC will be delivered via a patch, a topical cream, or in vaporizer or e-cigarette form.
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The central role of tRNA in genetic code expansion

The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation.
SCOPE OF REVIEW:
Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins.
MAJOR CONCLUSIONS:
The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio.
GENERAL SIGNIFICANCE:
Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions.
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CRISPR/Cas9 Reveals Cancer’s Synthetic Lethal Vulnerabilities

CRISPR/Cas9 Reveals Cancer’s Synthetic Lethal Vulnerabilities | SynBioFromLeukipposInstitute | Scoop.it
The CRISPR/Cas9 gene-editing system has been used to identify more than 120 synthetic-lethal gene interactions in cancer cells. These interactions could guide drug developers to new combination therapies that could selectively kill cancer cells and spare healthy cells.

Synthetic-lethal gene interactions may occur when certain pairs of mutated genes are present. When there is a mutation in either of these genes within a cell, the cell remains viable. But when there are mutations in both genes, the result is cell death. Synthetic-lethal gene interactions are especially important in the context of cancer therapies. If at least one of the genes in the interaction is specific to cancer, then a drug that inhibits the other gene would selectively kill only cancer cells.

The synthetic-lethal concept has been around for years, but it has been underdeveloped because chemical and genetic tools for the perturbation of gene function in somatic cells have been lacking. But this limitation has been addressed by researchers at the University of California, San Diego. These researchers report that they developed a new method to search for synthetic-lethal gene combinations.

The method appeared March 20 in the journal Nature Methods, in an article entitled “Combinatorial CRISPR–Cas9 Screens for De Novo Mapping of Genetic Interactions.”

“We developed a systematic approach to map human genetic networks by combinatorial CRISPR–Cas9 perturbations coupled to robust analysis of growth kinetics,” wrote the article’s authors. “We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction.”

In this article, the UC San Diego team described how they used the gene-editing technique CRISPR/Cas9 to simultaneously test for thousands of synthetic-lethal interactions. The researchers designed a CRISPR/Cas9 system with two guide RNAs: (1) one that targets a tumor suppressor gene that is commonly mutated in cancer and (2) one that targets a gene that could also be disrupted by a cancer drug. They deployed this system against 73 genes in three laboratory cell lines—human cervical cancer, lung cancer, and embryonic kidney cells. Then they measured cell growth and death.

“Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision,” the authors noted. “From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.”

"The ovarian cancer drug olaparib works by synthetic lethality—it inhibits a gene that, when a BRCA gene is also mutated, kills just those cancer cells," said John Paul Shen, M.D., clinical instructor and postdoctoral fellow at UC San Diego School of Medicine and Moores UCSD Cancer Center. "Many other cancers could likely be treated this way as well, but we don't yet know which gene mutation combinations will be synthetic-lethal."

"Identifying underlying genetic interactions in this way can reveal important functional relationships between genes, such as contributions to the same protein complex or pathway," co-senior author Trey Ideker, Ph.D., professor in the UC San Diego School of Medicine, founder of the UC San Diego Center for Computational Biology and Bioinformatics and co-director of the Cancer Cell Map Initiative. "This in turn can impact both our fundamental understanding of biological systems, as well as therapeutics development."

Many of the gene interactions the team identified were synthetic-lethal in just one of the three cell lines tested. This means that synthetic-lethal interactions may be different in different types of cancer. The researchers said this will be an important consideration for future drug development.

"Moving forward, we intend to further refine our technology platform and make it more robust," said co-senior author Prashant Mali, Ph.D., assistant professor in the Jacobs School of Engineering at UC San Diego. "And we are scaling our cancer genetic networks maps so we can systematically identify new combination therapies."
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Scientists Hack a Human Cell and Reprogram It Like a Computer

Scientists Hack a Human Cell and Reprogram It Like a Computer | SynBioFromLeukipposInstitute | Scoop.it
CELLS ARE BASICALLY tiny computers: They send and receive inputs and output accordingly. If you chug a Frappuccino, your blood sugar spikes, and your pancreatic cells get the message. Output: more insulin.

But cellular computing is more than just a convenient metaphor. In the last couple of decades, biologists have been working to hack the cells’ algorithm in an effort to control their processes. They’ve upended nature’s role as life’s software engineer, incrementally editing a cell’s algorithm—its DNA—over generations. In a paper published today in Nature Biotechnology, researchers programmed human cells to obey 109 different sets of logical instructions. With further development, this could lead to cells capable of responding to specific directions or environmental cues in order to fight disease or manufacture important chemicals.

Their cells execute these instructions by using proteins called DNA recombinases, which cut, reshuffle, or fuse segments of DNA. These proteins recognize and target specific positions on a DNA strand—and the researchers figured out how to trigger their activity. Depending on whether the recombinase gets triggered, the cell may or may not produce the protein encoded in the DNA segment.

A cell could be programmed, for example, with a so-called NOT logic gate. This is one of the simplest logic instructions: Do NOT do something whenever you receive the trigger. This study’s authors used this function to create cells that light up on command. Biologist Wilson Wong of Boston University, who led the research, refers to these engineered cells as “genetic circuits.”

Here’s how it worked: Whenever the cell did contain a specific DNA recombinase protein, it would NOT produce a blue fluorescent protein that made it light up. But when the cell did not contain the enzyme, its instruction was DO light up. The cell could also follow much more complicated instructions, like lighting up under longer sets of conditions.

Wong says that you could use these lit up cells to diagnose diseases, by triggering them with proteins associated with a particular disease. If the cells light up after you mix them with a patient’s blood sample, that means the patient has the disease. This would be much cheaper than current methods that require expensive machinery to analyze the blood sample.

Now, don’t get distracted by the shiny lights quite yet. The real point here is that the cells understand and execute directions correctly. “It’s like prototyping electronics,” says biologist Kate Adamala of the University of Minnesota, who wasn’t involved in the research. As every Maker knows, the first step to building complex Arduino circuits is teaching an LED to blink on command.

Pharmaceutical companies are teaching immune cells to be better cancer scouts using similar technology. Cancer cells have biological fingerprints, such as a specific type of protein. Juno Therapeutics, a Seattle-based company, engineers immune cells that can detect these proteins and target cancer cells specifically. If you put logic gates in those immune cells, you could program the immune cells to destroy the cancer cells in a more sophisticated and controlled way.

Programmable cells have other potential applications. Many companies use genetically modified yeast cells to produce useful chemicals. Ginkgo Bioworks, a Boston-based company, uses these yeast cells to produce fragrances, which they have sold to perfume companies. This yeast eats sugar just like brewer’s yeast, but instead of producing alcohol, it burps aromatic molecules. The yeast isn’t perfect yet: Cells tend to mutate as they divide, and after many divisions, they stop working well. Narendra Maheshri, a scientist at Ginkgo, says that you could program the yeast to self-destruct when it stops functioning properly, before they spoil a batch of high-grade cologne.

Wong’s group wasn’t the first to make biological logic gates, but they’re the first to build so many with consistent success. Of the 113 circuits they built, 109 worked. “In my personal experience building genetic circuits, you’d be lucky if they worked 25 percent of the time,” Wong says. Now that they’ve gotten these basic genetic circuits to work, the next step is to make the logic gates work in different types of cells.

But it won’t be easy. Cells are incredibly complicated—and DNA doesn’t have straightforward “on” and “off” switches like an electronic circuit. In Wong’s engineered cells, you “turn off” the production of a certain protein by altering the segment of DNA that encodes its instructions. It doesn’t always work, because nature might have encoded some instructions in duplicate. In other words: It’s hard to debug 3 billion years of evolution.
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Scientists turn mammalian cells into complex biocomputers

Scientists turn mammalian cells into complex biocomputers | SynBioFromLeukipposInstitute | Scoop.it
Computer hardware is getting a softer side. A research team has come up with a way of genetically engineering the DNA of mammalian cells to carry out complex computations, in effect turning the cells into biocomputers. The group hasn’t put those modified cells to work in useful ways yet, but down the road researchers hope the new programming techniques will help improve everything from cancer therapy to on-demand tissues that can replace worn-out body parts.

Engineering cells to function like minicomputers isn’t new. As part of the growing field of synthetic biology, research teams around the globe have been manipulating DNA for years to make cells perform simple actions like lighting up when oxygen levels drop. To date, most such experiments have been done in Escherichia coli and other bacteria, because their genes are relatively easy to manipulate. Researchers have also managed to link multiple genetic circuits together within a single cell to carry out more complex calculations in bacteria.

Scientists have tried to extend this to mammalian cells to create genetic circuitry that can help detect and treat human diseases. But efforts to construct large-scale genetic circuits in mammalian cells have largely failed: For complex circuits to work, the individual components—the turning on and off of different genes—must happen consistently. The most common way to turn a gene on or off is by using proteins called transcription factors that bind to and regulate the expression of a specific gene. The problem is these transcription factors “all behave slightly differently,” says Wilson Wong, a synthetic biologist at Boston University.
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How to Train a Cell-Cutting-Edge Molecular Tools

In biological systems, the formation of molecular complexes is the currency for all cellular processes. Traditionally, functional experimentation was targeted to single molecular players in order to understand its effects in a cell or animal phenotype. In the last few years, we have been experiencing rapid progress in the development of ground-breaking molecular biology tools that affect the metabolic, structural, morphological, and (epi)genetic instructions of cells by chemical, optical (optogenetic) and mechanical inputs. Such precise dissection of cellular processes is not only essential for a better understanding of biological systems, but will also allow us to better diagnose and fix common dysfunctions. Here, we present several of these emerging and innovative techniques by providing the reader with elegant examples on how these tools have been implemented in cells, and, in some cases, organisms, to unravel molecular processes in minute detail. We also discuss their advantages and disadvantages with particular focus on their translation to multicellular organisms for in vivo spatiotemporal regulation. We envision that further developments of these tools will not only help solve the processes of life, but will give rise to novel clinical and industrial applications.
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Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells | SynBioFromLeukipposInstitute | Scoop.it
Genetic circuits that are reliable, robust, and scalable are built without the need for optimization using a recombinase-based system.
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Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds | SynBioFromLeukipposInstitute | Scoop.it
Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, “green” technology for regenerating large volume vascularized tissue mass.
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20n: Computational synthetic biology

20n: Computational synthetic biology | SynBioFromLeukipposInstitute | Scoop.it
20n is open sourcing its platform for bioengineering
Over the last 4 years we have developed a better way to bioengineer organisms. We are now open sourcing our entire software stack, 20n/act. Find it at https://github.com/20n/act.

The stack will enumerate all bio-accessible chemicals, called reachables (20n/act/reachables). For each of those chemicals, it will design DNA blueprints. These DNA blueprints can bioengineering organisms with un-natural function. E.g., build organisms to make chemicals that were previously only sourced through petrochemistry.

To do that, the stack contains many modules built from scratch in-house. Some of them: mine raw biochemical data, integrate heterogenous sources, learn rules of biochemistry, automatically clean bad data, mine patents, mine plain text, bioinformatic identification of enzymes with desired function.

Once the suggested DNA is used to create new engineered cells those cells can be analyzed with LCMS for function. Our deep learning-based untargeted metabolomics stack processes the raw data and enumerates all side-effects of the changed genomic structure of the cell. Some would be expected, as the organism making the desired chemical, and some unexpected metabolic changes are highlighted.

We are also releasing a economic cost model for bioproduction. This economic cost model maps the desired the market price of the biological product to the "science needed" to get there. The "science needed" is measured in fermentation metrics, yield, titers, productivities of the engineered organisms. From that one can draw estimates of cost of investment (time and money) needed, and the expected ROI. More on this in a later blog post.

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Paper-based maskless enzymatic sensor for glucose determination combining ink and wire electrodes

In this work we have developed an amperometric enzymatic biosensor in a paper-based platform with a mixed electrode configuration: carbon ink for the working electrode (WE) and metal wires (from a low-cost standard electronic connection) for reference (RE) and auxiliary electrodes (AE). A hydrophobic wax-defined paper area was impregnated with diluted carbon ink. Three gold-plated pins of the standard connection are employed, one for connecting the WE and the other two acting as RE and AE. The standard connection works as a clip in order to support the paper in between. As a proof-of-concept, glucose sensing was evaluated. The enzyme cocktail (glucose oxidase, horseradish peroxidase and potassium ferrocyanide as mediator of the electron transfer) was adsorbed on the surface. After drying, glucose solution was added to the paper, on the opposite side of the carbon ink. It wets RE and AE, and flows by capillarity through the paper contacting the carbon WE surface. The reduction current of ferricyanide, product of the enzymatic reaction, is measured chronoamperometrically and correlates to the concentration of glucose. Different parameters related to the bioassay were optimized, adhering the piece of paper onto a conventional screen-printed carbon electrode (SPCE). In this way, the RE and the AE of the commercial card were employed for optimizing the paper-WE. After evaluating the assay system in the hybrid paper-SPCE cell, the three-electrode system consisting of paper-WE, wire-RE and wire-AE, was employed for glucose determination, achieving a linear range between 0.3 and 15 mM with good analytical features and being able of quantifying glucose in real food samples.
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Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method

DNA assembly forms the cornerstone of modern synthetic biology. Despite the numerous available methods, scarless multi-fragment assembly of large plasmids remains challenging. Furthermore, the upcoming wave in molecular biological automation demands a rethinking of how we perform DNA assembly. To streamline automation workflow and minimize operator intervention, a non-enzymatic assembly method is highly desirable. Here, we report the optimization and operationalization of a process called Twin-Primer Assembly (TPA), which is a method to assemble polymerase chain reaction-amplified fragments into a plasmid without the use of enzymes. TPA is capable of assembling a 7 kb plasmid from 10 fragments at ∼80% fidelity and a 31 kb plasmid from five fragments at ∼50% fidelity. TPA cloning is scarless and sequence independent. Even without the use of enzymes, the performance of TPA is on par with some of the best in vitro assembly methods currently available. TPA should be an invaluable addition to a synthetic biologist's toolbox.
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Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes

Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes | SynBioFromLeukipposInstitute | Scoop.it
Structured Abstract
INTRODUCTION
Controlling the spatial arrangement of functional components in biological systems on the scale of higher-order macromolecular assemblies is an important goal in synthetic biology. Achieving this goal could yield new research tools and pave the way for interesting applications in health and biotechnology. DNA origami enables constructing arbitrary shapes on the desired scale by folding a single-stranded DNA “scaffold” into user-defined shapes using a set of “staple” oligonucleotides, but single-stranded DNA is typically not available in living cells, and the structures usually assemble only at nonphysiological temperatures. The scope of functions that can be fulfilled by DNA itself appears so far still limited. Proteins, on the other hand, offer a large variety of functionalities and are easily accessible in cells through genetic encoding, but designing larger structural frameworks from proteins alone remains challenging.
RATIONALE
Here, we reimagined DNA origami to explore the possibility for using a set of designed proteins to fold a double-stranded DNA template into user-defined DNA-protein hybrid objects with dimensions on the desired 10- to 100-nm scale. To realize our idea, we require synthetic DNA “looping” proteins that can link two user-defined double-helical DNA sequences, and we need to determine suitable rules for arranging both the template and multiple of such staple proteins in the context of a larger target structure. Transcription activator–like (TAL) effector proteins are produced by plant pathogenic bacteria and injected into host cells, where they bind to specific promoter regions, thus controlling the expression of target genes. The DNA recognizing part of a TAL effector consists of an array of repeat subunits that binds to the major groove of double-helical DNA, thus forming a superhelix. Each repeat subunit comprises ~34 amino acids and recognizes a single DNA base pair. Because of their modular architecture, TAL effectors can be engineered to bind to user-defined DNA sequences, and this technique is currently being exploited for genome engineering applications. For constructing the staple proteins, we therefore chose the DNA-recognizing domains of TAL effectors.
RESULTS
We characterized the DNA-recognizing domains of the TAL effectors with respect to binding affinity and sequence specificity. To construct the staple proteins, we fused two TAL proteins via a custom peptide linker and tested for the ability to connect two separate double-helical DNA domains. For creating larger objects containing multiple staple protein connections, we identified a set of rules regarding the optimal spacing between these connections. On the basis of these rules, we could create megadalton-scale objects that realize a variety of structural motifs, such as custom curvatures, vertices, and corners. Each of those objects was built from a set of 12 double-TAL staple proteins and a template DNA double strand with designed sequence. We also tested design principles for multilayer structures with enhanced rigidity. All components of our nanostructures can be genetically encoded and self-assemble isothermally at room temperature in near-physiological buffer conditions. The staple proteins used in this work also carried a green fluorescent protein domain that serves as a placeholder for a variety of functional protein domains that can be genetically fused to the staple proteins. We were also able to demonstrate formation of our structures starting from genetic expression in a one-pot reaction mixture that contained the double-stranded DNA scaffold, the genes encoding the staple proteins, RNA polymerase, ribosomes, and cofactors for transcription and translation. Successful self-assembly of our hybrid nanostructures was confirmed using transmission electron microscopy.
CONCLUSION
By using our system of designing double-TAL staple proteins that fold a template DNA double strand, researchers can control the spatial arrangement of protein domains in custom geometries. Our system should have a good chance to work inside cells, given the success of our in vitro expression experiments and considering that the DNA binding properties of TAL are preserved inside cells, as seen in gene editing experiments with TAL-based endonucleases. Because TAL-based staple proteins can be tailored to specifically recognize any desired DNA target sequence, these proteins could then be used to create custom structures and loops in genomic DNA to study the relation between genome architecture and gene expression, or to position proteins involved in other intracellular processes in user-defined ways.
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New strategies and approaches for engineering biosynthetic gene clusters of microbial natural products

With the rapidly growing number of sequenced microbial (meta)genomes, enormous cryptic natural product (NP) biosynthetic gene clusters (BGCs) have been identified, which are regarded as a rich reservoir for novel drug discovery. A series of powerful tools for engineering BGCs has accelerated the discovery and development of pharmaceutically active NPs. Here, we describe recent advances in the strategies for BGCs manipulation, which are driven by emerging technologies, including efficient DNA recombination systems, versatile CRISPR/Cas9 genome editing tools and diverse DNA assembly methods. We further discuss how these approaches could be used for genome mining studies and industrial strain improvement.
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Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10 µg ml−1, as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20–40 min following exposure to 3 µg ml−1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera.
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