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Artificial Riboswitch Selection: A FACS-Based Approach

Artificial Riboswitch Selection: A FACS-Based Approach | SynBioFromLeukipposInstitute | Scoop.it
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Zohaib Ghazi, Casey C. Fowler, Yingfu Li

"Riboswitches have a number of characteristics that make them ideal regulatory elements for a wide range of synthetic biology applications. To maximize their utility, methods are required to create custom riboswitches de novo or to modify existing riboswitches to suit specific experimental needs. This chapter describes such a method, which exploits fluorescence-activated cell sorting (FACS) to quickly and efficiently sort through large libraries of riboswitch-like sequences to identify those with the desired activity. Suggestions for the experimental setup are provided, along with detailed protocols for testing and optimizing FACS conditions FACS selection steps, and follow-up assays to identify and characterize individual riboswitches."

http://bit.ly/1bqtoZa

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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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EmptPhage engineering: how advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophagesy title

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Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942

Cyanobacteria are oxygenic photosynthetic prokaryotes that are able to assimilate CO2 using solar energy and water. Metabolic engineering of cyanobacteria has suggested the possibility of direct CO2 conversion to value-added chemicals. However, engineering of cyanobacteria has been limited due to the lack of various genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for biochemicals from CO2. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942 as a model cyanobacterium. The SyneBrick chromosomal integration vectors provide three inducible expression systems to control gene expression and three neutral sites for chromosomal integrations. Using a SyneBrick vector, LacI-regulated gene expression led to 24-fold induction of the eYFP reporter gene with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer in S. elongatus PCC 7942 under 5% (v/v) CO2. TetR-regulated gene expression led to 19-fold induction of the GFP gene when 100 nM anhydrotetracycline (aTc) inducer was used. Gene expression decreased after 48 h due to degradation of aTc under light. T7 RNA polymerase-based gene expression resulted in efficient expression with a lower IPTG concentration than a previously developed pTrc promoter. A library of T7 promoters can be used for tunable gene expression. In summary, SyneBrick vectors were developed as a synthetic biology platform for gene expression in S. elongatus PCC 7942. These results will accelerate metabolic engineering of biosolar cell factories through expressing and controlling multiple genes of interest.
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Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.
RESULTS:
In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.
CONCLUSION:
Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.
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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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Bacteria Are Brewing Up the Next Generation of Antivenoms

Bacteria Are Brewing Up the Next Generation of Antivenoms | SynBioFromLeukipposInstitute | Scoop.it
DANIEL DEMPSEY WAS a grad student stationed in the jungles of Monteverde, Costa Rica when he first encountered the danger of a snakebite. The biologist was walking through the forest one day, catching bats to study them for malaria, when he almost stepped on the black, arrow-shaped head of an enormous pit viper—a fer-de-lance. That night as he described his encounter to the local family he was staying with, they began to tear up. They told him that earlier that year a “terciopelo,” what Costa Ricans call their country’s deadliest snake, had bitten the family’s five year-old niece. The hospital, a few hours drive away, didn’t have any antivenom in their stocks. She didn’t make it.

It was Dempsey’s memory of that little girl that made him leave his job as an antibody researcher at cancer pharmaceutical company, Celgene, to focus full-time on antivenoms. Every year, at least a hundred thousand people die from a run-in with one of the 375 venomous species of snake. And right now there’s a global shortage of the only thing that can save a bite victim: antivenom. For close to 100 years, antivenom production has been a laborious process of snake-milking and horse blood harvesting. But now, with synthetic biology and next-generation sequencing techniques, scientists are pushing the field into the future. Along with education and smart distribution, those advances could help end this global public health crisis.

A number of new biotech startups, both in the US and Europe, are signing on to tackle the antivenom problem. Dempsey’s, called Venomyx, is using vats of antibody-burping bacteria to engineer their cure. In a communal basement lab in San Francisco’s Tenderloin neighborhood, Dempsey and his employees Deepankar Roy and Alex Capovilla share bench, fridge, and instrument space to develop their pipeline of four antivenoms.

The first thing you might notice about the lab, if you know anything about antivenom production, is the distinct absence of farm animals. For decades, scientists have injected horses, or sometimes sheep, with a diluted version of snake venom, then collected their blood after a period of incubation and immune system triggering. Manufacturers use chemicals like ammonium sulfate or molecular separation methods to purify the antibodies. Then they suspend them in liquid and voila: antivenom. But to create their antibodies, Dempsey’s team is trading the equine incubator for the workhorse of the synthetic biology world: E. Coli, which they genetically modified to produce the venom-fighting stuff.

First, they injected a llama with sub-lethal amounts of snake venom. After sequencing the DNA of her antibody-creating B cells, they built a library of all the molecules those genes encoded. Then they exposed the library to tons of toxins found in snake venom—and after seeing which toxins stuck, they picked the tightest-binding molecules, and stuck their genes inside E. coli. The bacteria, when put in a bioreactor with the right mix of media and other molecules, kick out a soup of antibodies, which will go into different antivenoms for Asia, Africa, North America, and South America. Dempsey says that because llama antibodies are roughly 80 percent similar to the ones humans make, they don’t set off damaging immune responses, which can happen with horse and sheep-derived products.

But camelids aren’t the only new idea in town. A different startup, Copenhagen-based VenomAB, is instead using “humanized” antibodies to build out its antivenom product pipeline, which it has been developing with a Swedish pharmaceutical manufacturer. That process, which is popular in the cancer treatment world, involves designing human-like antibodies with variable regions that can bind different toxins, and getting bacteria to belch them out, just like they do for human insulin and other recombinant drug therapies. Both companies say that avoiding herds of hundreds or thousands of large mammals will bring down the time it takes to make the serums, as well as the cost. Typical doses can cost between $800 and $1,000 in rural Africa, and up to $14,000 in the US.

Solving the antivenom shortage, of course, is more complicated than simply developing more efficient production lines. The vast majority of mortalities fall on poor, isolated communities like subsistence farmers in Sub-Saharan Africa and Southeast Asia, so big manufacturers in the US and Europe have been slow to get into the game. In that vacuum, companies in Asia and India have flooded the market with subpar products, that might not even be applicable to places like Africa that have totally different species of snakes. The other big problem is that most of the antivenoms currently on the market require refrigeration. Developing countries, with their spotty infrastructure, needs shelf-stable serums that can survive tropical temperatures. “Good antivenoms can be made really affordably—$14 or $20 a vial,” says Leslie Boyer, founding director of the University of Arizona’s VIPER Institute. “But it’s the distribution costs that make the situation untenable.”

These new approaches to antivenom development are valuable from a research standpoint. But they may not be necessary to solve the global shortage. “Do we need the most cutting edge technologies modern science can offer?” says Boyer. “No. What we need is better distribution networks, and certification programs to regulate the quality of the products and education programs to build trust with communities.” To that end, she and her colleagues in Arizona are teaming up with experts from Mexico and Africa to launch an international awareness campaign to areas hardest hit by snakebites.

Dempsey is hoping that in a few years from now, when his shelf-stable, horse-free antivenoms are ready for prime time, the efforts of people like Boyer will make it easier to get treatments to people where they need it most. Places like the jungles of the Congo and the mountains of Costa Rica.
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Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics

Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics | SynBioFromLeukipposInstitute | Scoop.it
Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics.
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Scooped by Gerd Moe-Behrens
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Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells

The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca2+). By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca2+-activated processes.
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Supramolecular Hydrogels Based on DNA Self-Assembly

Extracellular matrix (ECM) provides essential supports three dimensionally to the cells in living organs, including mechanical support and signal, nutrition, oxygen, and waste transportation. Thus, using hydrogels to mimic its function has attracted much attention in recent years, especially in tissue engineering, cell biology, and drug screening. However, a hydrogel system that can merit all parameters of the natural ECM is still a challenge. In the past decade, deoxyribonucleic acid (DNA) has arisen as an outstanding building material for the hydrogels, as it has unique properties compared to most synthetic or natural polymers, such as sequence designability, precise recognition, structural rigidity, and minimal toxicity. By simple attachment to polymers as a side chain, DNA has been widely used as cross-links in hydrogel preparation. The formed secondary structures could confer on the hydrogel designable responsiveness, such as response to temperature, pH, metal ions, proteins, DNA, RNA, and small signal molecules like ATP. Moreover, single or multiple DNA restriction enzyme sites could be incorporated into the hydrogels by sequence design and greatly expand the latitude of their responses. Compared with most supramolecular hydrogels, these DNA cross-linked hydrogels could be relatively strong and easily adjustable via sequence variation, but it is noteworthy that these hydrogels still have excellent thixotropic properties and could be easily injected through a needle. In addition, the quick formation of duplex has also enabled the multilayer three-dimensional injection printing of living cells with the hydrogel as matrix. When the matrix is built purely by DNA assembly structures, the hydrogel inherits all the previously described characteristics; however, the long persistence length of DNA structures excluded the small size meshes of the network and made the hydrogel permeable to nutrition for cell proliferation. This unique property greatly expands the cell viability in the three-dimensional matrix to several weeks and also provides an easy way to prepare interpenetrating double network materials. In this Account, we outline the stream of hydrogels based on DNA self-assembly and discuss the mechanism that brings outstanding properties to the materials. Unlike most reported hydrogel systems, the all-in-one character of the DNA hydrogel avoids the "cask effect" in the properties. We believe the hydrogel will greatly benefit cell behavior studies especially in the following aspects: (1) stem cell differentiation can be studied with solely tunable mechanical strength of the matrix; (2) the dynamic nature of the network can allow cell migration through the hydrogel, which will help to build a more realistic model to observe the migration of cancer cells in vivo; (3) combination with rapidly developing three-dimension printing technology, the hydrogel will boost the construction of three-dimensional tissues and artificial organs.
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