Publisher Correction: Modular and tunable biological feedback control using a de novo protein switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Modular and tunable biological feedback control using a de novo protein switch.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

The pGinger Family of Expression Plasmids.

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of Gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of red fluorescent protein (RFP), with a broad-host-range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry. IMPORTANCE Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria.