The Coli Toolkit (CTK): An Extension of the Modular Yeast Toolkit for Use in <i>E. coli</i>

Genetic circuits are a cornerstone of synthetic biology, enabling programmable control of cellular behavior for applications in health, sustainability, and biotechnology. While Genetic Design Automation (GDA) tools have optimized and streamlined the design of such circuits, rapid and efficient assembly of DNA remains a bottleneck in the Design-Build-Test-Learn (DBTL) cycle. Here, we present the Coli Toolkit (CTK), a modular Golden Gate-based cloning system, adapted from the Yeast Toolkit (YTK) for use in <i>Escherichia coli</i>. The CTK expands on the original YTK architecture by introducing a more flexible control of transcription and translation through subdividing the former promoter part into subparts: promoter, insulating ribozyme, and ribosome binding site (RBS). We provide a range of basic parts that enable the assembly of a wide range of constructs as well as characterization data for all constitutive and inducible promoters provided. Additionally, we provide characterization data, as well as calibrated models, for all 20 NOT gates from the Cello library, and we provide the NOT gates as preassembled basic parts, which enables rapid cloning of larger genetic circuits. With this toolkit, we leverage the strengths of the YTK architecture to enable rapid and high-efficiency assembly of genetic circuits in <i>E. coli</i>, filling a key gap in the infrastructure of bacterial synthetic biology.