A MoClo-compatible Toolbox of ECF Sigma Factor-Based Regulatory Switches for Proteobacterial Chassis

The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components. Previously, extracytoplasmic function sigma factors (ECFs), a diverse group of alternative sigma factors with distinct promoter specificities, were shown to have great potential as context-independent regulators, but so far, they have only been used in a few model species. Here, we show that the alphaproteobacterium Sinorhizobium meliloti, which has been proposed as a plant-associated bacterial chassis for synthetic biology, has a similar phylogenetic ECF acceptance range as the gammaproteobacterium Escherichia coli. A common set of orthogonal ECF-based regulators that can be used in both bacterial hosts was identified and used to create 2-step delay circuits. The genetic circuits were implemented in single copy in E. coli by chromosomal integration using an established method that utilizes bacteriophage integrases. In S. meliloti, we demonstrated the usability of single-copy pABC plasmids as equivalent carriers of the synthetic circuits. The circuits were either implemented on a single pABC or modularly distributed on 3 such plasmids. In addition, we provide a toolbox containing pABC plasmids compatible with the Golden Gate (MoClo) cloning standard and a library of basic parts that enable the construction of ECF-based circuits in S. meliloti and in E. coli. This work contributes to building a context-independent and species-overarching ECF-based toolbox for synthetic biology applications.