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

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