Contextual dependencies expand the re-usability of genetic inverters

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.

Refactoring of a synthetic raspberry ketone pathway with EcoFlex

“…In summary, these results confirmed that the characterisation of individual gene-fusions differ in relation to enzyme function and the state of protein folding within the cell. This is a form of context dependency, which is an increasingly important factor in the design of genetic circuits and pathways in synthetic biology [ 23 ]. Next, with this new knowledge, we set upon refactoring the pathway to optimise raspberry ketone production.…”

Functional biology in its natural context: A search for emergent simplicity

“…Will they keep their function over evolutionary timescales, which are quite short in cellular populations? Ecological robustness and evolutionary stability should be part of the design of genetic circuits ( Cardinale and Arkin, 2012 ; Tas et al, 2021 ). Likewise, efforts to ‘edit’ the microbiome in situ should contemplate the ecological and evolutionary response of the community to those genetic changes ( Brenner et al, 2009 ; Chang et al, 2021 ; Goldman and Brown, 2009 ; McCarty and Ledesma-Amaro, 2019 ).…”

Interrogation of Essentiality in the Reconstructed Haemophilus influenzae Metabolic Network Identifies Lipid Metabolism Antimicrobial Targets: Preclinical Evaluation of a FabH β-Ketoacyl-ACP Synthase Inhibitor

“…In addition, the high quality of i NL638, in terms of consistency and interoperability, paves the way for the future multistrain modeling of the H. influenzae species using the strain RdKW20 as a reference. As shown by the strain-dependent effect of the inhibitor tested, the impact of metabolic context is gaining relevance as a key factor driving cell behavior ( 67 ). Therefore, further H. influenzae multistrain modeling effort could lead to the identification of secondary metabolic hubs modulating the response of known and novel drugs, promoting the establishment of synergistic and more robust antibacterial treatments.…”