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

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.