Abstract
The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. Although these are mostly focussed on the genetic components alone, the context in which the circuit performs is crucial for its outcome. We characterise 20 genetic NOT logic gates in up to 7 bacterial-based contexts each, to generate 135 different functions. The contexts we focus on are combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each gate shows seven different dynamic behaviours, depending on the context. That is, gates can be fine-tuned by changing only contextual parameters, thus improving the compatibility between gates. Finally, we analyse portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expands functionality, and advocate for considering context as a fundamental design parameter.
Highlights
The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology
To generate enough data on the contextual dependencies of genetic inverters we made use of 20 NOT logic gates assembled with a suite of promoters and repressors first developed as components of the CELLO platform for E. coli[1] and recloned in broad host range vectors of different copy numbers for delivery to different types Gramnegative hosts[23]
In order to manipulate the expression level of the regulator, its coding sequence was placed under the control of a lac promoter, which was externally induced by IPTG
Summary
The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. A core objective of synthetic biology[3] is the building of new regulatory circuits to compute inputs into outputs according to predefined logical functions[4], which are used in a number of applications, ranging from bioproduction[5] to medical diagnosis[6] This approach has been relatively successful, genetic logic gates are far more fragile and less reliable than their electronic counterparts as their signals are rarely constant and often fluctuate over time[7,8]. A fundamental challenge for the design of robust synthetic circuits, which underpins this work, is the oversimplified model that assumes DNA elements (i.e., gates) alone explain the performance of genetic circuits Based on this assumption, the host chassis (the cell that receives a specific genetic construct) is generally ignored and the interplay of a genetic circuit with the host context is most often overlooked in the bottom-up design process—an issue that has been identified as essential for the predictability of synthetic biology devices[12]. We propose to utilise a strategy that is inspired by nature, and includes context as a parameter in the design of optimal genetic circuits
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