Antagonistic Control of Genetic Circuit Performance for Rapid Analysis of Targeted Enzyme Activity in Living Cells.

Kil Koang Kwon,Eugene Rha,Haseong Kim,Soo-Jin Yeom,Seung-Goo Lee,Hyewon Lee,Dae-Hee Lee,Jinju Lee

doi:10.3389/fmolb.2020.599878

Abstract

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise criterion for enzyme screening and engineering. Antagonists and inhibitors are small molecules with inhibitory effects on regulators and enzymes, respectively. In this study, an antagonist and an inhibitor were applied to a genetic circuit for a dynamic detection range. We developed a genetic circuit relying on regulators and enzymes, allowing for straightforward control of its output signal without additional genetic modification. We used para-nitrophenol and alanine as an antagonist of DmpR and inhibitor of tyrosine phenol-lyase, respectively. We show that the antagonist resets the detection range of the genetic circuit similarly to a resistor in an electrical logic circuit. These biological resistors in genetic circuits can be used as a rapid and precise controller of variable outputs with minimal circuit configuration.

Highlights

Designing a genetic circuit is a crucial step for programming of living organisms, a long-term aim of synthetic biology (Brophy and Voigt, 2014; Nielsen et al, 2016)
To detect enzymatic activity arising from the antagonistic effect of pNP, cells harboring pDmpR-genetic enzyme screening system (GESS) and the tyrosine phenol-lyase (TPL) gene were grown in lysogeny broth (LB) with 20 μM L-rhamnose, 10 μM pyridoxal 5′-phosphate (PLP), 50 μg/mL ampicillin, and 25 μg/mL chloramphenicol
Addition of an enzyme inhibitor and a regulator antagonist can act as resistors for the two AND gates

Summary

INTRODUCTION

Designing a genetic circuit is a crucial step for programming of living organisms, a long-term aim of synthetic biology (Brophy and Voigt, 2014; Nielsen et al, 2016). Fine tuning of gene expression is necessary to enable complex and precise control of biological reactions This fine tuning can be achieved by adjusting the output signal range of the genetic elements (Brophy and Voigt, 2014; Smanski et al, 2014). These molecules can bind to the active site of proteins and decrease their activity by interfering with enzymesubstrate or regulator-ligand complex formation This property can be used to control the sensitivity and dynamic range of a genetic circuit by adding small molecules to the reaction system (Xie et al, 2014). By adjusting the concentration of the small molecules, they can be used as variable resistors for tunable signal production These biological resistors may be implemented for switchable and precise monitoring of enzyme activity, while the genetic circuit maintains hypersensitivity without additional genetic modification. In various applications, such as enzyme evolution, the circuit could be controlled using bio-parts and related inhibitors or antagonists as GCRs

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSIONS

DATA AVAILABILITY STATEMENT