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Bayesian design strategies for synthetic biology

Bayesian design strategies for synthetic biology | SynBioFromLeukipposInstitute | Scoop.it
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by
Barnes CP, Silk D, Stumpf MP.

"We discuss how statistical inference techniques can be applied in the context of designing novel biological systems. Bayesian techniques have found widespread application and acceptance in the systems biology community, where they are used for both parameter estimation and model selection. Here we show that the same approaches can also be used in order to engineer synthetic biological systems by inferring the structure and parameters that are most likely to give rise to the dynamics that we require a system to exhibit. Problems that are shared between applications in systems and synthetic biology include the vast potential spaces that need to be searched for suitable models and model parameters; the complex forms of likelihood functions; and the interplay between noise at the molecular level and nonlinearity in the dynamics owing to often complex feedback structures. In order to meet these challenges, we have to develop suitable inferential tools and here, in particular, we illustrate the use of approximate Bayesian computation and unscented Kalman filtering-based approaches. These partly complementary methods allow us to tackle a number of recurring problems in the design of biological systems. After a brief exposition of these two methodologies, we focus on their application to oscillatory systems."

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Bioinformatics Software for Synthetic Biology

Bioinformatics Software for Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it
Archetype® software is a one-of-a-kind solution that enables researchers to discover, analyze and build synthetic genes and pathways.
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Cells programmed like computers to fight disease

Cells programmed like computers to fight disease | SynBioFromLeukipposInstitute | Scoop.it
Cells can be programmed like a computer to fight cancer, influenza, and other serious conditions – thanks to a breakthrough in synthetic biology by the University of Warwick.
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Frontiers | Image of Synthetic Biology and Nanotechnology: A Survey Among University Students

Frontiers | Image of Synthetic Biology and Nanotechnology: A Survey Among University Students | SynBioFromLeukipposInstitute | Scoop.it
This study explores the image of synthetic-biology and nanotechnology in comparison to agricultural biotechnology and communication-technology by examining spontaneous associations with, and deliberate evaluations of, these technologies by university students. Data were collected through a self-completion online questionnaire by students from two universities in Switzerland. The survey aimed to capture implicit associations, explicit harm-benefit-evaluations and views on regulation. The data suggest overall positive associations with emerging technologies. While positive associations were most pronounced for nanotechnology, agricultural biotechnology was attributed with the least favourable associations. In contrast to its positive result in the association task, respondents attributed a high harm potential for nanotechnology. Associations attributed to synthetic-biology were demonstrated to be more positive than for agricultural biotechnology, however, not as favourable as for nanotechnology. Contrary to the evaluations of nanotechnology, the benefit-examples of synthetic-biology were evaluated particularly positively. Accordingly, the investigated technologies enjoy different esteem, with synthetic biology and nanotechnology both showing a more “exciting” image. Even though the image of nanotechnology was demonstrated to be more pronounced it was also more heterogeneous across tasks while agricultural biotechnology remains contested. For all technologies, the predominant spontaneous concerns pertain to risks rather than an immoral nature inherent to these technologies. Our data suggest that harm-benefit-analyses reveal only one aspect of the attitude towards emerging technologies. Survey questions addressing spontaneous associations with these technologies are a valuable addition to our picture of the image of emerging technologies.
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A cargo-sorting DNA robot

Sorting molecules with DNA robots
Single-stranded DNA robots can move over the surface of a DNA origami sheet and sort molecular cargoes. Thubagere et al. developed a simple algorithm for recognizing two types of molecular cargoes and their drop-off destinations on the surface (see the Perspective by Reif). The DNA robot, which has three modular functional domains, repeatedly picks up the two types of molecules and then places them at their target destinations. No additional power is required because the DNA robot does this by random walking across the origami surface.
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Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors

Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors | SynBioFromLeukipposInstitute | Scoop.it
Using a highly programmable split-dCas9-based signal transduction module, Baeumler
et al. have created a novel class of synthetic receptors (dCas9-synRs) capable of
coupling biologically relevant input signals with the direct activation of custom
user-defined output response programs. dCas9-synRs expand the promise of cellular
engineering for research and therapeutic applications.
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Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing—the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product—varies in a target site–dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.
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Course: Introduction to Synthetic Biology - New York

Course: Introduction to Synthetic Biology - New York | SynBioFromLeukipposInstitute | Scoop.it
Eventbrite - SynBioBeta presents Course: Introduction to Synthetic Biology - New York - Friday, September 15, 2017 at 7 West 34th Street, New York, NY. Find event and ticket information.
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Modified viruses deliver death to antibiotic-resistant bacteria

Modified viruses deliver death to antibiotic-resistant bacteria | SynBioFromLeukipposInstitute | Scoop.it
Engineered microbes turn a bacterium's immune response against itself using CRISPR.
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Semiconductor nanoparticles give bacteria a boost

Semiconductor nanoparticles give bacteria a boost | SynBioFromLeukipposInstitute | Scoop.it
Bacteria covered in semiconductor nanoparticles make more efficient use of light and could be used to create a key ingredient in fuel and plastics.
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Native extracellular matrix-derived semipermeable, optically transparent, and inexpensive membrane inserts for microfluidic cell culture

Native extracellular matrix-derived semipermeable, optically transparent, and inexpensive membrane inserts for microfluidic cell culture | SynBioFromLeukipposInstitute | Scoop.it
Semipermeable cell culture membranes are commonly used in multilayered microfluidic devices to mimic the basement membrane in vivo and to create compartmentalized microenvironments for physiological cell growth and differentiation. However, existing membranes are predominantly made up of synthetic polymers, providi
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Droplet microfluidics for synthetic biology

Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to enable the possibility of evaluating thousands of gene variants and hundreds of thousands of gene combinations. However, such large-scale optimization experiments remain cost-prohibitive to researchers following traditional molecular biology practices, which are frequently labor-intensive and suffer from poor reproducibility. Liquid handling robotics may reduce labor and improve reproducibility, but are themselves expensive and thus inaccessible to most researchers. Microfluidic platforms offer a lower entry price point alternative to robotics, and maintain high throughput and reproducibility while further reducing operating costs through diminished reagent volume requirements. Droplet microfluidics have shown exceptional promise for synthetic biology experiments, including DNA assembly, transformation/transfection, culturing, cell sorting, phenotypic assays, artificial cells and genetic circuits.
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Droplet Microfluidics for Synthetic Biology

Droplet Microfluidics for Synthetic Biology | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to enable the possibility of evaluating thousands of gene variants and hundreds of thousands of gene combinations. However, such large-scale optimization experiments remain cost-prohibitive to researchers following traditional molecular biology practices, which are frequently labor-intensive and suffer from poor reproducibility. Liquid handling robotics may reduce labor and improve reproducibility, but are themselves expensive and thus inaccessible to most researchers. Microfluidic platforms offer a lower entry price point alternative to robotics, and maintain high throughput and reproducibility while further reducing operating costs through diminished reagent volume requirements. Droplet microfluidics have shown exceptional promise for synthetic biology experiments, including DNA assembly, transformation/transfection, culturing, cell sorting, phenotypic assays, artificial cells and genetic circuits.
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Soft robotics: self-contained soft actuator three times stronger than natural muscle, without the need of externals

Soft robotics: self-contained soft actuator three times stronger than natural muscle, without the need of externals | SynBioFromLeukipposInstitute | Scoop.it
Researchers at Columbia Engineering have solved a long-standing issue in the creation of untethered soft robots whose actions and movements can help mimic natural biological systems. A group in the Creative Machines lab le
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Designer Bacteria as Intratumoural Enzyme Biofactories

Bacterial-directed enzyme prodrug therapy (BDEPT) is an emerging form of treatment for cancer. It is a biphasic variant of gene therapy in which a bacterium, armed with an enzyme that can convert an inert prodrug into a cytotoxic compound, induces tumour cell death following tumour-specific prodrug activation. BDEPT combines the innate ability of bacteria to selectively proliferate in tumours, with the capacity of prodrugs to undergo contained, compartmentalised conversion into active metabolites in vivo. Although BDEPT has undergone clinical testing, it has received limited clinical exposure, and has yet to achieve regulatory approval. In this article, we review BDEPT from the system designer's perspective, and provide detailed commentary on how the designer should strategize its development de novo. We report on contemporary advancements in this field which aim to enhance BDEPT in terms of safety and efficacy. Finally, we discuss clinical and regulatory barriers facing BDEPT, and propose promising approaches through which these hurdles may best be tackled.
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Structures of the CRISPR genome integration complex

Host factor drives the big bend
Bacteria have a highly adaptable DNA-detecting and -editing machine called CRISPR-Cas to ward off virus attack. The Cas1-Cas2 integrase, with the help of an accessory protein called IHF (integration host factor), captures foreign DNA motifs into bacterial CRISPR loci. These motifs then act as sensors of any further invaders. By analyzing the integrase complex structure, Wright et al. show how Cas1-Cas2 recognizes the CRISPR array for site-specific integration (see the Perspective by Globus and Qimron). IHF sharply bends DNA, which allows DNA to access two active sites within the integrase complex to ensure sequence specificity for the integration reaction. The features of the CRISPR integrase complex may explain the natural divergence of CRISPR arrays in bacteria and can be exploited for genome-tagging applications.
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MIT Media Lab's Journal of Design and Science Is a Radical New Kind of Publication

MIT Media Lab's Journal of Design and Science Is a Radical New Kind of Publication | SynBioFromLeukipposInstitute | Scoop.it
The MIT Media Lab has launched a new kind of academic journal that embodies its "antidisciplinary" ethos.
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Signal transduction in a covalent post-assembly modification cascade

Signal transduction in a covalent post-assembly modification cascade | SynBioFromLeukipposInstitute | Scoop.it
Natural reaction cascades control the movement of biomolecules between cellular compartments. Inspired by these systems, we report a synthetic reaction cascade employing post-assembly modification reactions to direct the partitioning of supramolecular complexes between phases. The system is composed of a self-assembled tetrazine-edged FeII8L12 cube and a maleimide-functionalized FeII4L6 tetrahedron. Norbornadiene (NBD) functions as the stimulus that triggers the cascade, beginning with the inverse-electron-demand Diels–Alder reaction of NBD with the tetrazine moieties of the cube. This reaction generates cyclopentadiene as a transient by-product, acting as a relay signal that subsequently undergoes a Diels–Alder reaction with the maleimide-functionalized tetrahedron. Cyclooctyne can selectively inhibit the cascade by outcompeting NBD as the initial trigger. Initiating the cascade with 2-octadecyl NBD leads to selective alkylation of the tetrahedron upon cascade completion. The increased lipophilicity of the C18-tagged tetrahedron drives this complex into a non-polar phase, allowing its isolation from the initially inseparable mixture of complexes.
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Designing randomized DNA sequences free of restriction enzyme recognition sites

DNA libraries containing random "barcodes" complicate synthetic biology workflows that utilize restriction enzymes, since restriction sites can appear inside some barcodes. By removing bases at particular sites in the barcodes, it is possible to create semi-random pools of barcodes that do not contain any restriction sites. The challenge is to remove as few bases as possible to maximize the number of sequences in the pool while ensuring all sequences are free of restriction sites. We present CutFree, a computational approach to create pools of random DNA barcodes that lack a pre-defined set of restriction sites. The resulting pools can be inexpensively produced en masse with standard DNA synthesis techniques. We experimentally validated CutFree by blocking digestion of pools of barcodes designed to frequently contain restriction sites. Using CutFree, we designed a pool of 1.3 billion barcodes that are free from recognition sites for 182 commercially-available restriction enzymes. CutFree is available as a software package and an online tool
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Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers

Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers | SynBioFromLeukipposInstitute | Scoop.it
In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
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The timing of transcriptional regulation in synthetic gene circuits

Transcription factors and their target promoters are central to synthetic biology. By arranging these components into novel gene regulatory circuits, synthetic biologists have been able to create a wide variety of phenotypes, including bistable switches, oscillators, and logic gates. However, transcription factors (TFs) do not instantaneously regulate downstream targets. After the gene encoding a TF is turned on, the gene must first be transcribed, the transcripts must be translated, and sufficient TF must accumulate in order to bind operator sites of the target promoter. The time to complete this process, here called the "signaling time," is a critical aspect in the design of dynamic regulatory networks, yet it remains poorly characterized. In this work, we measured the signaling time of two TFs in Escherichia coli commonly used in synthetic biology: the activator AraC and the repressor LacI. We found that signaling times can range from a few to tens of minutes, and are affected by the expression rate of the TF. Our single-cell data also show that the variability of the signaling time increases with its mean. To validate these signaling time measurements, we constructed a two-step genetic cascade, and showed that the signaling time of the full cascade can be predicted from those of its constituent steps. These results provide concrete estimates for the timescales of transcriptional regulation in living cells, which are important for understanding the dynamics of synthetic transcriptional gene circuits.
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Droplet microfluidics for synthetic biology 

Droplet microfluidics for synthetic biology  | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to ena
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Protein design: I like to fold it, fold it

Protein design: I like to fold it, fold it | SynBioFromLeukipposInstitute | Scoop.it
Most proteins have evolved for function rather than stability, which makes it difficult to ascertain the connections between sequence and folding and, in turn, confounds de novo protein design. Combining the expression of synthetic DNA libraries with proteolysis resistance assays, Rocklin et al. have now systematically determined the stability of over 15,000 designed miniproteins in a high-throughput fashion. Following the screen, each sequence was assigned a 'stability score', and a subset was validated by structural analyses including circular dichroism and NMR
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Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo

Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo | SynBioFromLeukipposInstitute | Scoop.it
Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.
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