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Gerd Moe-Behrens
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Mammalian synthetic biology: emerging medical applications

by
Zoltán Kis , Hugo Sant'Ana Pereira , Takayuki Homma , Ryan M. Pedrigi , Rob Krams

"In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes."

http://bit.ly/1CU8GN0
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Gerd Moe-Behrens
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Scientists Urge Temporary Moratorium On Human Genome Edits

by
Rob Stein

"A new technology called CRISPR could allow scientists to alter the human genetic code for generations. That's causing some leading biologists and bioethicists to sound an alarm. They're calling for a worldwide moratorium on any attempts to alter the code, at least until there's been time for far more research and discussion.

Jennifer Doudna and her colleagues found an enzyme in bacteria that makes editing DNA in animal cells much easier.

In Hopes Of Fixing Faulty Genes, One Scientist Starts With The Basics
The CRISPR enzyme (green and red) binds to a stretch of double-stranded DNA (purple and red), preparing to snip out the faulty part.

It's not new that scientists can manipulate human DNA — genetic engineering, or gene editing, has been around for decades. But it's been hard, slow and very expensive. And only highly skilled geneticists could do it.

Recently that's changed. Scientists have developed new techniques that have sped up the process and, at the same time, made it a lot cheaper to make very precise changes in DNA.

There are a couple of different techniques, but the one most often talked about is CRISPR, which stands for clustered regularly interspaced short palindromic repeats. My colleague Joe Palca described the technique for Shots readers last June.

Why scientists are nervous

On the one hand, scientists are excited about these techniques because they may let them do good things, such as discovering important principles about biology. It might even lead to cures for diseases.

The big worry is that CRISPR and other techniques will be used to perform germline genetic modification...."

http://n.pr/1Fuyn8T
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Gerd Moe-Behrens
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Programmable RNA recognition and cleavage by CRISPR/Cas9

by
Mitchell R. O’Connell, Benjamin L. Oakes, Samuel H. Sternberg, Alexandra East-Seletsky, Matias Kaplan & Jennifer A. Doudna

"The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1, 2, 3, 4, 5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4, 5, 6, 7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags."

http://bit.ly/13kZBNX
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Gerd Moe-Behrens
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New device could make large biological circuits practical

by
David L. Chandler 

"Researchers have made great progress in recent years in the design and creation of biological circuits — systems that, like electronic circuits, can take a number of different inputs and deliver a particular kind of output. But while individual components of such biological circuits can have precise and predictable responses, those outcomes become less predictable as more such elements are combined.
A team of researchers at MIT has now come up with a way of greatly reducing that unpredictability, introducing a device that could ultimately allow such circuits to behave nearly as predictably as their electronic counterparts. The findings are published this week in the journal Nature Biotechnology, in a paper by associate professor of mechanical engineering Domitilla Del Vecchio and professor of biological engineering Ron Weiss.
The lead author of the paper is Deepak Mishra, an MIT graduate student in biological engineering. Other authors include recent master’s students Phillip Rivera in mechanical engineering and Allen Lin in electrical engineering and computer science.
There are many potential uses for such synthetic biological circuits, Del Vecchio and Weiss explain. “One specific one we’re working on is biosensing — cells that can detect specific molecules in the environment and produce a specific output in response,” Del Vecchio says. One example: cells that could detect markers that indicate the presence of cancer cells, and then trigger the release of molecules targeted to kill those cells.
It is important for such circuits to be able to discriminate accurately between cancerous and noncancerous cells, so they don’t unleash their killing power in the wrong places, Weiss says. To do that, robust information-processing circuits created from biological elements within a cell become “highly critical,” Weiss says.
To date, that kind of robust predictability has not been feasible, in part because of feedback effects when multiple stages of biological circuitry are introduced. The problem arises because unlike in electronic circuits, where one component is physically connected to the next by wires that ensure information is always flowing in a particular direction, biological circuits are made up of components that are all floating around together in the complex fluid environment of a cell’s interior.
Information flow is driven by the chemical interactions of the individual components, which ideally should affect only other specific components. But in practice, attempts to create such biological linkages have often produced results that differed from expectations.
“If you put the circuit together and you expect answer ‘X,’ and instead you get answer ‘Y,’ that could be highly problematical,” Del Vecchio says.
The device the team produced to address that problem is called a load driver, and its effect is similar to that of load drivers used in electronic circuits: It provides a kind of buffer between the signal and the output, preventing the effects of the signaling from backing up through the system and causing delays in outputs.
While this is relatively early-stage research that could take years to reach commercial application, the concept could have a wide variety of applications, the researchers say. For example, it could lead to synthetic biological circuits that constantly measure glucose levels in the blood of diabetic patients, automatically triggering the release of insulin when it is needed.
The addition of this load driver to the arsenal of components available to those designing biological circuits, Del Vecchio says, “could escalate the complexity of circuits you could design,” opening up new possible applications while ensuring that their operation is “robust and predictable.”
James Collins, a professor of biomedical engineering at Boston University who was not associated with this research, says, “Efforts in synthetic biology to create complex gene circuits are often hindered by unanticipated or uncharacterized interactions between submodules of the circuits. These interactions alter the input-output characteristics of the submodules, leading to undesirable circuit behavior.”..."


http://bit.ly/1pOFToz
Innovation from MIT could allow many biological components to be connected to produce predictable effects.
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Gerd Moe-Behrens
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Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells 

by
Yuchen Liu, Yayue Zeng, Li Liu, Chengle Zhuang, Xing Fu, Weiren Huang & Zhiming Cai

"The conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. A possible way to overcome these limitations is to construct logic circuits. Here we present modular AND gate circuits based on CRISPR-​Cas9 system. The circuits integrate cellular information from two promoters as inputs and activate the output gene only when both inputs are active in the tested cell lines. Using the luciferase reporter as the output gene, we show that the circuit specifically detects bladder cancer cells and significantly enhances luciferase expression in comparison to the human ​telomerase reverse transcriptase-renilla luciferase construct. We also test the modularity of the design by replacing the output with other cellular functional genes including ​hBAX, ​p21 and ​E-cadherin. The circuits effectively inhibit bladder cancer cell growth, induce apoptosis and decrease cell motility by regulating the corresponding gene. This approach provides a synthetic biology platform for targeting and controlling bladder cancer cells in vitro."


http://bit.ly/1xqfP2b
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Gerd Moe-Behrens
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Accurate Predictions of Genetic Circuit Behavior from Part Characterization and Modular Composition

by
Noah Davidsohn , Jacob Beal , Samira Kiani , Aaron Adler , Fusun Yaman , Yinqing Li , Zhen Xie , and Ron Weiss

"A long-standing goal of synthetic biology is to rapidly engineer new regulatory circuits from simpler devices. As circuit complexity grows, it becomes increasingly important to guide design with quantitative models, but previous efforts have been hindered by lack of predictive accuracy. To address this, we developed Empirical Quantitative Incremental Prediction (EQuIP), a new method for accurate prediction of genetic regulatory network behavior from detailed characterizations of their components. In EQuIP, precisely calibrated time-series and dosage-response assays are used to construct hybrid phenotypic/mechanistic models of regulatory processes. This hybrid method ensures that model parameters match observable phenomena, using phenotypic formulation where current hypotheses about biological mechanisms do not agree closely with experimental observations. We demonstrate EQuIP’s precision at predicting distributions of cell behaviors for six transcriptional cascades and three feed-forward circuits in mammalian cells. Our cascade predictions have only 1.6-fold mean error over a 261-fold mean range of fluorescence variation, owing primarily to calibrated measurements and piecewise-linear models. Predictions for three feed-forward circuits had a 2.0-fold mean error on a 333-fold mean range, further demonstrating that EQuIP can scale to more complex systems. Such accurate predictions will foster reliable forward engineering of complex biological circuits from libraries of standardized devices."


 http://bit.ly/10YVNSk
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Gerd Moe-Behrens
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Technology of the future: Self-assembling is new 4D mantra

by
Anonymous

"3D printing has not yet got into the mainstream but we already have new advanced 4D printing technology that can revolutionise the way things to be printed in the future.

Imagine a day when you can build structures using mushrooms, a complete aircraft cabins with self-adjusting seats and space that can change according to the users’ requirement, a car that can self-assemble itself, a Nano robot that can get into your body and release just the required amount of payload of medication necessary to heal.

Work is on to create the new 4D printing technology, more advanced and scientific that it can self-assemble different parts and create a complete solution.

The technology is still under study at primary stages of testing, mainly at the Massachusetts Institute of Technology (MIT) and led by researcher Skylar Tibbits and Carlos Olguin, Head of Bio Nano Programmable Matter Group at Autodesk.

Tibbits is working on bio molecular self-assembly and human scale 4D printing and printing multi-materials that can change its shape over time.

As part of his Project Cyborg Tibbits used to simulate various shape transformations and optimise folding sequences in 4D Printing, the video below is a demonstration of Tibbits research at the TED conference in 2013...."


http://bit.ly/10vMzwC
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Gerd Moe-Behrens
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Rational Design of a Fusion Protein to Exhibit Disulfide-Mediated Logic Gate Behavior

by
Jay H. Choi and Marc Ostermeier 

"Synthetic cellular logic gates are primarily built from gene circuits owing to their inherent modularity. Single proteins can also possess logic gate functions and offer the potential to be simpler, quicker, and less dependent on cellular resources than gene circuits. However, the design of protein logic gates that are modular and integrate with other cellular components is a considerable challenge. As a step toward addressing this challenge, we describe the design, construction, and characterization of AND, ORN, and YES logic gates built by introducing disulfide bonds into RG13, a fusion of maltose binding protein and TEM-1 β-lactamase for which maltose is an allosteric activator of enzyme activity. We rationally designed these disulfide bonds to manipulate RG13’s allosteric regulation mechanism such that the gating had maltose and reducing agents as input signals, and the gates could be toggled between different gating functions using redox agents, although some gates performed sub optimally."

http://bit.ly/1FdmUIV
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Gerd Moe-Behrens
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Digital switching in a biosensor circuit via programmable timing of gene availability

by
Nicolas Lapique & Yaakov Benenson

"Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup."

 http://bit.ly/1o9lvhg
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Gerd Moe-Behrens
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Colloidal nanoparticles as advanced biological sensors

by
Philip D. Howes, Rona Chandrawati, Molly M. Stevens

"Nanoparticle biosensors have the potential to enhance or supersede current analytical techniques, and their introduction could have a great impact in research and clinical practice. The unique optical properties of many nanomaterials make them ideal for biosensing. Colloidal fluorescent and plasmonic nanoparticles are particularly interesting, as they produce intense responses to incident light, and linking this response to the presence of a target analyte yields extremely sensitive detection in solution."

 http://bit.ly/1xXcZ8d
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Gerd Moe-Behrens
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THE BIOLOGICAL MICROPROCESSOR http://flip.it/FuN9v via @flipboard
By Gerd Moe-Behrens | Systemics, a revolutionary paradigm shift in scientific thinking, with applications in systems biology, and synthetic biology, have led to the idea of using silicon computers and their engineering principles as a blueprint for the engineering of a similar machine made from biological parts....
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Gerd Moe-Behrens
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A prudent path forward for genomic engineering and germline gene modification

by
David Baltimore Paul Berg, Michael Botchan, Dana Carroll, R. Alta Charo, George Church, Jacob E. Corn, George Q. Daley, Jennifer A. Doudna Marsha Fenner, Henry T. Greely, Martin Jinek, G. Steven Martin, Edward Penhoet, Jennifer Puck, Samuel H. Sternberg, Jonathan S. Weissman, Keith R. Yamamoto

"A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed"

http://bit.ly/1BnD398
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Gerd Moe-Behrens
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Programmable materials and the nature of the DNA bond

by
Matthew R. Jones, Nadrian C. Seeman2, Chad A. Mirkin

"BACKGROUND
Nucleic acids are ubiquitous in biology because of their ability to encode vast amounts of information via canonical Watson-Crick base-pairing interactions. With the advent of chemical methods to make synthetic oligonucleotides of an arbitrary sequence, researchers can program entire libraries of molecules with orthogonal interactions, directed to assemble in highly specific arrangements. Early attempts to use DNA to make nanostructures led to topologically defined architectures, but ones that were too conformationally flexible to be used to guide the construction of well-defined nanoscale materials from the bottom up. In this Review, we discuss the key discoveries that have overcome this limitation and distill common design principles that have since led to a revolution in materials sophistication based on DNA-directed assembly.

ADVANCES
The experimental realization of DNA-based constructs that are sufficiently rigid so as to impart directionality to hybridization interactions marks a major milestone in the development of programmable materials assembly. This feat was accomplished simultaneously by the Mirkin Group and Seeman Group in 1996, but through chemically and conceptually distinct pathways. In one approach, rigidity is derived from multiple strand crossover events and the hybridization that stabilizes them to create a conformationally restricted DNA tile. In the other approach, a rigid non-nucleic acid–based nanoparticle (inorganic or organic) core acts as a template to organize functionalized DNA strands in a surface-normal orientation. It is appealing to draw the analogy between DNA-based constructs of this sort with the concepts of “bonds” and “valency” found in atomic systems. Just as understanding the nature of atomic bonding is crucial for chemists to manipulate the formation of molecular and supramolecular species, so too is an understanding of the nature of these DNA bonding modes necessary for nanoscientists to build complex and functional architectures to address materials needs.

OUTLOOK
The interest in nanoscale materials constructed by using DNA bonds has continued to grow steadily, but has seen a noteworthy explosion in relevance over the past several years. This is due in large part to the development of methods to move beyond simple clusters and crystals to more sophisticated nanostructured materials that are dynamic and stimuli responsive, are macroscopic in spatial extent, and exhibit emergent physical properties that arise from specific arrangements of matter. These techniques offer perhaps the most versatile way of organizing optically active materials into architectures that exhibit unusual and deliberately tailorable plasmonic and photonic properties. In addition, prospects include the use of these materials in biological settings, being that they are constructed, in large measure, from nucleic acid precursors. The ability to manipulate gene expression, deliver molecular payloads via DNA-based binding events, and detect relevant markers of disease with nanoscale spatial resolution represent some of the most fruitful avenues of future research."

http://bit.ly/1EyPgeD
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Gerd Moe-Behrens
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Synthetic biology: Toehold gene switches make big footprints

by
Simon Ausländer & Martin Fussenegger

"The development of RNA-based devices called toehold switches that regulate translation might usher in an era in which protein production can be linked to almost any RNA input and provide precise, low-cost diagnostics."


http://bit.ly/1DRsNNN
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Gerd Moe-Behrens
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Human thoughts used to switch on genes 
 http://bit.ly/1yA7vNy

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Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant

by
Marc Folcher, Sabine Oesterle, Katharina Zwicky, Thushara Thekkottil, Julie Heymoz, Muriel Hohmann, Matthias Christen, Marie Daoud El-Baba, Peter Buchmann & Martin Fussenegger


"Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain–computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein ​SEAP (​secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger ​cyclic diguanosine monophosphate (​c-di-GMP), which triggers the ​stimulator of interferon genes (​STING)-dependent induction of synthetic ​interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control ​SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice."

 http://bit.ly/110e0ON
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Gerd Moe-Behrens
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Can Bio-Computers Kill Cancer Cells

by
Chuck Seegert

"New research from ETH Zurich has made progress towards achieving bio-computers by developing a biological circuit that controls individual sensory components. Future developments of this technology could enable complex, cancer-hunting bio-computers.

Creating logic-based circuits from biological components has been challenging because of the nature of neural cells and how they interact. While an electronic computer is based on the discrete on and off signals that ones and zeros represent, biological circuits are much less predictable. Action potentials, the primary signal type that is transmitted by neural circuits, are subject to many influences, making signal transfer more unpredictable.

A reliable computer must be predictable, which is what researchers from ETH Zurich (Eidgenössische Technische Hochschule Zürich) may have done for biological computer systems. The technology may soon come to a point where a bio-computer could be feasible, according to a recent press release from the university.

“The ability to combine biological components at will in a modular, plug-and-play fashion means that we now approach the stage when the concept of programming as we know it from software engineering can be applied to biological computers,” said Yaakov Benenson, professor of synthetic biology in the Department of Biosystems Science and Engineering at ETH Zurich in Basel, in the press release. “Bio-engineers will literally be able to program in future.”

The technology combines genetics and signals from a special enzyme — called a recombinase — to activate a biological sensor only when it is signaled to do so. Essentially, the active gene is installed in the biosensor’s DNA in the wrong orientation, which makes it inactive, according to the press release. When a recombinase enzyme is put in the cellular environment, the gene is reoriented into the proper position, making the circuit active.

The new method enables sensors with a dynamic range that is up to 1,000-fold that of the originally configured systems, according to study published by the team in Nature Chemical Biology. The team foresees that this technology may be able to force cancer cells to undergo programmed cell death, while normal cells would remain inactivated.

While controlling the internal cellular machinery may be critical to generating bio-computers, directing and controlling where neurons grow may also be important in the design process. Directing neural cell growth in culture using fluid flow was recently discussed in an article published on Med Device Online."



? http://bit.ly/1vPrqoW
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Gerd Moe-Behrens
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Just add waterprogrammable in vitro diagnostics

by
Linda Koch

"Concerns about biosafety, practicality and costs have traditionally confined engineered synthetic gene circuits hosted in living cells to research environments, despite the great potential that these molecular tools hold for biotechnology and medical applications. Now, US researchers have circumvented these difficulties by using porous materials such as paper as host substrates for cell-free synthetic…"


 http://bit.ly/1uBp8zi
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Gerd Moe-Behrens
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Opening the (Protein Logic) Gates to Better Sensing,
Identifying and Reporting of Infectious Agents

by
Ilya Elashvili

"Researchers at Johns Hopkins University have found a way to engineer protein logic gates that sense, identify, and report on the presence of infectious agents without causing harm to the warfighter. This basic research effort, managed by DTRA CB/JSTO’s Dr. Ilya Elashvili and performed by Johns Hopkins University’s Drs. Jay
H. Choi and Marc Ostermeier, describes proof of principle experiments that demonstrates the successful design of artificial fusion proteins that function as protein
logic gates. The introduction of these proteins into cells resulted in cells with the desired logic gate function.
Eventually, researchers hope to create autonomously functioning cells that would seek and destroy pathogens and possibly diagnose and cure maladies that could affect warfighters.
Synthetic biology seeks to build programmed cells that respond to molecular signals with a desired behavior. Two-input logic gates are a fundamental unit of decision-making and thus have been a focal point for building programmed cells from
the ground up. With a few notable exceptions, the predominant focus in synthetic biology has been on building transcriptional network logic gates that function on the DNA level, which are then combined to build more complex behavior. However, conceptually, protein logic gates offer advantages
of simplicity, speed, and reduced consumption of cellular resources....."

 http://bit.ly/1Dj350X

as a comment to this original paper:

Rational Design of a Fusion Protein to Exhibit Disulfide-Mediated Logic Gate Behavior
 http://bit.ly/1FdmUIV
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Gerd Moe-Behrens
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Environmental Sensing of Heavy Metals Through Whole Cell Microbial Biosensors: A Synthetic Biology Approach

by
Lara Bereza-Malcolm , Gulay Mann , and Ashley Edwin Franks

"Whole cell microbial biosensors are offering an alternative means for rapid, on-site heavy metal detection. Based in microorganisms, biosensing constructs are designed and constructed to produce both qualitative and quantitative outputs in response to heavy metal ions. Previous microbial biosensors designs are focused on single-input constructs; however development of multiplexed systems is resulting in more flexible designs. The movement of microbial biosensors from laboratory based designs towards on-site, functioning heavy metal detectors has been hindered by the toxic nature of heavy metals, along with the lack of specificity of heavy metals promoter elements. Applying a synthetic biology approach with alternative microbial chassis may increase the robustness of microbial biosensors and mitigate these issues. Before full applications are achieved, further consideration has to be made regarding the risk and regulations of whole cell microbial biosensor use in the environment. To this end, a standard framework for future whole cell microbial biosensor design and use is proposed."


 http://bit.ly/1r3EmEY
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Gerd Moe-Behrens
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Protein-responsive ribozyme switches in eukaryotic cells

by
Kennedy AB, Vowles JV, d'Espaux L, Smolke C

"Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers."


http://bit.ly/1nXDE1v
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