Modular Golden Gate Assembly of linear DNA templates for cell-free prototyping

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

doi:10.1007/978-1-0716-4220-7_11

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Modular Golden Gate Assembly of linear DNA templates for cell-free prototyping

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Lehr, Francois-Xavier
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Gaizauskaite, Aukse
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Lipińska, Katarzyna Elżbieta
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Gilles, Sara
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Sahoo, Arpita
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Inckemann, René
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Niederholtmeyer, Henrike
Emmy Noether research Group Cell-free Synthetic Biology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;
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Citation

Lehr, F.-X., Gaizauskaite, A., Lipińska, K. E., Gilles, S., Sahoo, A., Inckemann, R., et al. (2025). Modular Golden Gate Assembly of linear DNA templates for cell-free prototyping. In D. Schindler (Ed.), Golden Gate Cloning. Methods in Molecular Biology (pp. 197-217). Humana, New York, NY. doi:10.1007/978-1-0716-4220-7_11.

Cite as: https://hdl.handle.net/21.11116/0000-000F-E7C9-A

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-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription 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 polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor–promoter pair. By eliminating time-consuming 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 assembled constructs within a single day.