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Ángeles Hueso-Gil1, Jesús Miró-Bueno1, Ángel Goñi-Moreno2

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Researchers engineered bacterial RNA polymerase (RNAP) for tunable growth control, enabling stable cellular states. This breakthrough offers new possibilities for biocomputation and synthetic biology applications.

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Systems Biology

Background:

  • Cellular context significantly impacts genetic circuit performance, posing challenges for rational control.
  • The interaction between host cells and engineered genetic circuits is complex and difficult to manipulate.
  • RNA polymerase (RNAP) activity is a key determinant of cellular state and gene expression.

Purpose of the Study:

  • To develop a method for rationally controlling genetic circuit performance by modulating the cellular context.
  • To engineer a tunable bacterial growth regulation system using an inducible RNA polymerase.
  • To explore the potential of growth control as an engineering parameter in synthetic biology.

Main Methods:

  • Replaced native RNA polymerase (RNAP) subunits (β and β') in *Pseudomonas putida* KT2440 with an inducible XylS-Pm system.
  • Utilized the inducer 3-methylbenzoate to tune bacterial cell growth into distinct stable states.
  • Correlated genetic circuit behavior (reporter gene expression, NOT gate performance) with cellular growth states.
  • Developed a mathematical model to classify RNAP availability's influence on host-circuit interactions.

Main Results:

  • Achieved tunable cell growth regulation, enabling distinct stable growth states.
  • Demonstrated that modulation of host-circuit dependencies is specific to circuit components.
  • Classified the impact of RNAP availability on host-circuit interactions into three categories via mathematical modeling.
  • Integrated growth control into a two-input NAND gate circuit, highlighting potential for morphological computing.

Conclusions:

  • Bacterial growth control can be rationally engineered as a parameter for optimizing biocomputation.
  • This approach has potential applications in microbial consortia engineering and the development of digital twins.
  • Modulating RNAP availability offers a novel strategy for controlling genetic circuit performance within a cellular context.