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Fluorescent fusion protein knockout mediated by anti-GFP nanobody - Nature Struct. Mol. Biology

Fluorescent fusion protein knockout mediated by anti-GFP nanobody - Nature Struct. Mol. Biology | About Synthetic Biology | Scoop.it

http://www.nature.com/nsmb/journal/v19/n1/full/nsmb.2180.html

Caussinus et al (2012)

The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.


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aamoros2's curator insight, June 24, 2014 12:39 PM

Proteínas fluorescentes.

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Genome-editing tools storm ahead : Nature Methods : Nature Publishing Group

Genome-editing tools storm ahead : Nature Methods : Nature Publishing Group | About Synthetic Biology | Scoop.it
The menu of maturing, diversifying methods calls for careful selections in experimental design.
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Iterative plug-and-play methodology for constructing and modifying synthetic gene networks

Iterative plug-and-play methodology for constructing and modifying synthetic gene networks | About Synthetic Biology | Scoop.it

Kevin D Litcofsky,Raffi B Afeyan,Russell J Krom,Ahmad S Khalil& James J Collins

 

Nature Methods9,1077–1080(2012)doi:10.1038/nmeth.2205

 

Abstract

 

We present a methodology for the design, construction and modification of synthetic gene networks. This method emphasizes post-assembly modification of constructs based on network behavior, thus facilitating iterative design strategies and rapid tuning and repurposing of gene networks. The ease of post-construction modification afforded by this approach and the ever-increasing repository of components within the framework will help accelerate the development of functional genetic circuits for synthetic biology.

 

http://www.nature.com/nmeth/journal/v9/n11/full/nmeth.2205.html?WT.ec_id=NMETH-201211

 

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Cell - A Whole-Cell Computational Model Predicts Phenotype from Genotype

Cell - A Whole-Cell Computational Model Predicts Phenotype from Genotype | About Synthetic Biology | Scoop.it

Jonathan R. Karr, Jayodita C. Sanghvi, Derek N. Macklin, Miriam V. Gutschow, Jared M. Jacobs, Benjamin Bolival, Nacyra Assad-Garcia, John I. Glass, Markus W. Covert

 

Cell, Volume 150, Issue 2, 389-401, 20 July 2012

 

Summary:

 

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery.

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Optical Control of Protein Activity by Fluorescent Protein Domains

Optical Control of Protein Activity by Fluorescent Protein Domains | About Synthetic Biology | Scoop.it

Science 9 November 2012:
Vol. 338 no. 6108 pp. 810-814

 

Xin X. Zhou, Hokyung K. Chung, Amy J. Lam, Michael Z. Lin

 

Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.

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An adaptor from translational to transcriptional control enables predictable assembly of complex regulation

An adaptor from translational to transcriptional control enables predictable assembly of complex regulation | About Synthetic Biology | Scoop.it

Chang C Liu,Lei Qi,Julius B Lucks,Thomas H Segall-Shapiro,Denise Wang,Vivek K Mutalik& Adam P Arkin

 

Nature Methods9,1088–1094(2012)doi:10.1038/nmeth.2184

 

Abstract:

 

Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule–triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

 

http://www.nature.com/nmeth/journal/v9/n11/full/nmeth.2184.html?WT.ec_id=NMETH-201211

 

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Genetic programs constructed from layered logic gates in single cells

Genetic programs constructed from layered logic gates in single cells | About Synthetic Biology | Scoop.it

Tae Seok Moon,Chunbo Lou,Alvin Tamsir,Brynne C. Stanton& Christopher A. Voigt

Nature491,249–253(08 November 2012)doi:10.1038/nature11516

Received02 February 2012Accepted15 August 2012Published online07 October 2012

 

Abstract:

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits1, 7. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

 

http://www.nature.com/nature/journal/v491/n7423/full/nature11516.html

 

 

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