Browsing by Author "Haseloff, Jim"

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    Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts
    (2021) Arce, Anibal; Guzman Chavez, Fernando; Gandini, Chiara; Puig, Juan; Matute, Tamara; Haseloff, Jim; Dalchau, Neil; Molloy, Jenny; Pardee, Keith; Federici, Fernan
    Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.
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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.