Browsing by Author "Pollak, Bernardo"
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- ItemLoop assembly: a simple and open system for recursive fabrication of DNA circuit(2019) Pollak, Bernardo; Cerda, Ariel; Delmans, Mihails; Álamos, Simón; Moyano, Tomás; West, Anthony; Gutiérrez, Rodrigo A.; Patron, Nicola J.; Federici, Fernán; Haseloff, JimHigh-efficiency methods for DNA assembly have enabled the routine assembly of syntheticDNAs of increased size and complexity. However, these techniques require customization,elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. Thesystem makes use of two Type IIS restriction endonucleases and corresponding vector sets forefficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assem-bled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets.The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. Weshow the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%).We have characterized Loop assembly on over 200 different DNA constructs and validatedthe fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalized solution for DNA construction with standardizedparts. The cloning system is provided under an OpenMTA license for unrestricted sharing andopen access.
- ItemUniversal loop assembly: open, efficient and cross-kingdom DNA fabrication.(2020) Pollak, Bernardo; Matute Torres, Tamara Francisca; Núñez Quijada, Isaac Natán; Cerda Rojas, Ariel; López Sierra, Constanza Andrea; Vargas Torres, Valentina Isabel; Kan, Anton; Bielinski,Vincent; Dassow, Peter von; Dupont, Chris L.; Federici, FernánStandardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a 'common syntax' that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in hostspecific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe 'universal Loop' (uLoop) assembly, a system based on Loop assembly for use in potentially any organismof choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.