# The Coli Toolkit (CTK): An Extension of the Modular Yeast Toolkit for Use in E. coli

## TL;DR

The Coli Toolkit (CTK) is a new cloning system for E. coli that improves the speed and efficiency of building genetic circuits.

## Contribution

CTK adapts the Yeast Toolkit for E. coli with modular parts and calibrated models for rapid circuit assembly.

## Key findings

- CTK introduces subdivided promoter parts for better control of transcription and translation.
- Characterization data and calibrated models are provided for 20 NOT gates from the Cello library.
- Preassembled parts enable faster construction of complex genetic circuits in E. coli.

## Abstract

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 Escherichia coli. 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 E. coli, filling
a key gap in the infrastructure of bacterial synthetic biology.