Browsing by Author "Patron, Nicola J."

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    Loop 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, Jim
    High-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.