A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria

Tas, Huseyin; Goñi-Moreno, Ángel

doi:10.1101/2020.07.14.202754

Cited by 2 publications

(4 citation statements)

References 17 publications

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“…Therefore, genetic logic gates are exposed to contextual dependencies that influence their phenotypic behaviour. In the extreme case, these constraints can even result in unviability of the cellular host, for example, during the conversion to pSEVA broad host range backbones, five of the genetic inverters were not functional when cloned into a high-copy plasmid (pSEVA251), perhaps due to overload in the allocation of cellular resources resulting in toxicity 23 .…”

Section: Discussionmentioning

confidence: 99%

“…The repressible and inducible systems were previously described by Voigt Lab and acquired by the courtesy of Voigt Laboratory in MIT (USA). Description of the 20 different NOT gates moved into broad host range pSEVA backbones are described in Tas et al 23 . Components of the original inverters, like terminators, RBSs and insulators were kept the same during the SEVA conversion.…”

Section: Methodsmentioning

confidence: 99%

“…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 then recloned in broad host range vectors of different copy numbers for delivery to different types Gramnegative hosts 23 . The logic function (NOT or inverter) corresponds to a genetic device that reverses the incoming signal (i.e.…”

Section: Generation Of Gate-context Librariesmentioning

confidence: 99%

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Contextual dependencies expand the re-usability of genetic inverters

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Generation Of Gate-context Librariesmentioning

confidence: 99%

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Contextual dependencies expand the re-usability of genetic inverters

et al. 2021

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“…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 then recloned in broad host range vectors of different copy numbers for delivery to different types Gram-negative hosts 23 . The logic function (NOT or inverter) corresponds to a genetic device that expresses a target gene (output high) if it is not negatively regulated (input low) and vice-versa.…”

Section: Generation Of Gate-context Librariesmentioning

confidence: 99%

Contextual dependencies expand the re-usability of genetic inverters

Tas

Grozinger

Stoof

et al. 2020

Preprint

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The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial for its outcome. Here, we characterise 20 genetic NOT logic gates (inverters) in up to 7 bacterial-based contexts each, to finally generate 135 different functions. The contexts we focus on are particular combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each NOT logic gate shows seven different logic behaviours, depending on the context. That is, gates can be reconfigured to fit response requirements by changing only contextual parameters. Computational analysis shows that this range of behaviours improves the compatibility between gates, because there are considerably more possibilities for combination than when considering a unique function per genetic construct. Finally, we address the issue of interoperability and 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 expand the scope of the functions that can be engineered in complex cellular environments, and advocate for considering context as a fundamental design parameter for synthetic biology.

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A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria

Cited by 2 publications

References 17 publications

Contextual dependencies expand the re-usability of genetic inverters

Contextual dependencies expand the re-usability of genetic inverters

Contextual dependencies expand the re-usability of genetic inverters

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