Synthetic Biological Circuits within an Orthogonal Central Dogma

Abstract

Synthetic biology strives to reliably control cellular behavior, typically in the form of user-designed interactions of biological components to produce a predetermined output. Engineered circuit components are frequently derived from natural sources and are therefore often hampered by inadvertent interactions with host machinery, most notably within the host central dogma. Reliable and predictable gene circuits require the targeted reduction or elimination of these undesirable interactions to mitigate negative consequences on host fitness and develop context-independent bioactivities. Here, we review recent advances in biological orthogonalization, namely the insulation of researcher-dictated bioactivities from host processes, with a focus on systematic developments that may culminate in the creation of an orthogonal central dogma and novel cellular functions. Engineering Gene Circuits Natural gene circuits (see Glossary) rely on regulatory factors to govern cellular activities, leveraging elements of both circuit insulation and interaction to afford the necessary responses [1]. These natural gene circuits are maintained through discrete functional modules, which allow for both robustness and plasticity in adapting to changing environmental conditions [2]. Over the past two decades, components of natural gene circuits have been isolated and repurposed to develop increasingly complex engineered gene circuits [3], wherein multiple user-defined inputs may act in concert to produce the desired outputs [4]. Increasing circuit complexity has been driven by the incremental realization of synthetic biology goals: controlled gene expression [5], genetic code expansion [6] and development of synthetic cells [7]. In turn, these and other accomplishments have catalyzed significant research into more complex genetic circuitry that continue to co-opt host cell machinery and resources for researcher-dictated bioactivities. Highlights Development of fully synthetic nucleobase pairs that faithfully interact in living cells, and their applications in creating semisynthetic organisms with expanded and orthogonal informationcarrying capacity.

Heterologous production of glycine betaine using Synechocystis sp. PCC 6803-based chassis lacking native compatible solutes

“…This design ensured the stable constitutive transcription observed for the GB device ORFs, regardless of the chassis or salinity conditions (Fig. 7), reinforcing that the use of orthogonal regulatory components is crucial to ensure the proper insulation of synthetic devices from the regulatory network of the chassis (54). Unlike transcription, the synthesis of the solute was not independent of cultivation conditions, and GB levels increased with salinity.…”

Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes

“…Besides the ORFs required for GB production (gsmt and dmt) and metX (to prevent SAM shortage), this device comprises well-characterized regulatory elements: the synthetic promoter P trc.x.lacO (Ferreira et al, 2018), the RBS BBa_B0030, and the double terminator BBa_B0015. This design ensured the stable constitutive transcription observed for the GB device ORFs, regardless of the chassis or salinity conditions (Figure 7), reinforcing that the use of orthogonal regulatory components is crucial to ensure the proper insulation of synthetic devices from the regulatory network of the chassis (Costello and Badran, 2021). Unlike transcription, the synthesis of the solute was not independent of cultivation conditions, and GB levels increased with salinity.…”

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkBdevice

“…Ideally, for engineering reliable genetic devices regulatory parts should deliver their function in a context-independent manner (29). Such a context includes not only genomic locations (30) and availability of resources (31), but also physiological signals (32,33) that orchestrate the induction hierarchy.…”