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

TL;DR

This paper presents a rapid, modular Golden Gate assembly method to produce linear DNA templates for cell-free systems, significantly speeding up genetic circuit prototyping without cloning steps.

Contribution

It introduces a Golden Gate-based workflow for quick assembly of linear DNA templates from modular parts for cell-free prototyping.

Findings

Rapid assembly of DNA templates within a single day

Elimination of cloning steps accelerates testing cycles

Compatible with modular cloning toolboxes

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.