Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Integração da simulação termoenergética nas primeiras fases do processo projetual: o estudo de seis casos

Resumo O artigo tem o objetivo de demonstrar a viabilidade de integração de simulações termoenergéticas ao processo projetual desde as primeiras fases do projeto. Para tanto, apresenta uma sistematização dos procedimentos entre projetista e simulador para seis estudos de caso de edificações em clima quente. Destaca-se a necessidade de compreensão das características do edifício e da programação arquitetônica, incluindo informações, metas e necessidade, além de partido e intenções da solução, que motivaram a montagem de um checklist para melhorar o diálogo entre simulador e projetista. Os resultados também apontam as características da edificação que mais influenciam no consumo energético da edificação, e a interação entre o simulador e o projetista, de acordo com seu perfil, as liberdades e restrições implícitas, e a recorrência das soluções, que poderiam até mesmo dispensar as simulações. Além da confirmação de que é possível integrar simulações desde as primeiras fases, a contribuição principal do artigo é o mapeamento de processos entre projetista e simulador e sua intensidade para demonstrar as potencialidades e limitações das integrações, que podem ser úteis no desenvolvimento de interfaces de ferramentas e no treinamento de especialistas em simulação

Energy-oriented design tools for collaboration in the cloud

Emerging from the challenge to reduce energy consumption in buildings is the need for energy simulation to be used more effectively to support integrated decision making in early design. As a critical response to a Green Star case study, we present DEEPA, a parametric modeling framework that enables architects and engineers to work at the same semantic level to generate shared models for energy simulation. A cloud-based toolkit provides web and data services for parametric design software that automate the process of simulating and tracking design alternatives, by linking building geometry more directly to analysis inputs. Data, semantics, models and simulation results can be shared on the fly. This allows the complex relationships between architecture, building services and energy consumption to be explored in an integrated manner, and decisions to be made collaboratively

Achieving informed decision-making for net zero energy buildings design using building performance simulation tools

Building performance simulation (BPS) is the basis for informed decision-making of Net Zero Energy Buildings (NZEBs) design. This paper aims to investigate the use of building performance simulation tools as a method of informing the design decision of NZEBs. The aim of this study is to evaluate the effect of a simulation-based decision aid, ZEBO, on informed decision-making using sensitivity analysis. The objective is to assess the effect of ZEBO and other building performance simulation tools on three specific outcomes: (i) knowledge and satisfaction when using simulation for NZEB design; (ii) users’ decision-making attitudes and patterns, and (iii) performance robustness based on an energy analysis. The paper utilizes three design case studies comprising a framework to test the use of BPS tools. The paper provides results that shed light on the effectiveness of sensitivity analysis as an approach for informing the design decisions of NZEBs