Modular Golden Gate Assembly of Linear DNA Templates for Cell-free Prototyping

François-Xavier Lehr; Aukse Gaizauskaite; Katarzyna Elżbieta Lipińska; Sara Gilles; Arpita Sahoo; René Inckemann; Henrike Niederholtmeyer

arXiv · 2310.13665

Modular Golden Gate Assembly of Linear DNA Templates for Cell-free Prototyping

François-Xavier Lehr·Aukse Gaizauskaite·Katarzyna Elżbieta Lipińska·Sara Gilles·Arpita Sahoo·René Inckemann·Henrike Niederholtmeyer

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Abstract

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test cycle of new functions in synthetic biology, in particular when linear DNA templates are used. Here we describe a Golden Gate assisted workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcriptional units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by PCR amplification. By eliminating lengthy transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new constructs within a single day.

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François-Xavier Lehr, Aukse Gaizauskaite, Katarzyna Elżbieta Lipińska, Sara Gilles, Arpita Sahoo, René Inckemann, Henrike Niederholtmeyer. 2023-10-20. Modular Golden Gate Assembly of Linear DNA Templates for Cell-free Prototyping. https://arxiv.org/abs/2310.13665

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