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Cells of Rhodosporidium toruloides CECT1137, grown on media with C/N ratio of (a) 10, (b) 20, (c) 30, (d) 60, (e) 90, (f) 120. Lipids are stained with BODIPY. Pictures were taken by microscopy, using a YFP fluorescence filter and a Nomarski illumination
Source publication
This protocol describes a single-cell high-throughput genetic enzyme screening system (GESS) in which GFP fluorescence is used to detect the production of phenolic compounds from a given substrate by metagenomic enzyme activity. One of the important features of this single-cell genetic circuit is that it can be used to screen more than 200 differen...
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Metagenomic datasets from pangolin tissue specimens have previously yielded SARS-related coronaviruses which show high homology in their receptor binding domain to SARS-CoV-2, suggesting a potential zoonotic source for this feature of the human virus, possibly via recombination (Liu et al. 2019, Lam et al. 2020, Xiao et al. 2020, Liu et al. 2020)....
Citations
... In this strategy, we previously showed that a phenyl moiety is a putative target in numerous enzyme substrates. 22 The phenyl moiety in artificial substrates has advantages such as intracellular stability; simple chemical synthesis; and a planar, compact, and rigid structure. Other structures of artificial substrates, except for phenyl moieties, can be designed for various functional groups considering the target enzyme activity. ...
... Many aromatic compounds can diffuse freely through the cell membrane via passive transport (Gallert and Winter, 1993;Chen and Fink, 2006). In addition, more than 200 enzymes can generate phenol or pNP from phenolic substrates through their catalytic reactions (Kim et al., 2015). Intermolecular release using phenolic substrates and heterologous enzymes in different cells can affect circuit signals in terms of pattern generation or edge detection. ...
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.
