Reconstruction of biological subsystems using bottom‐up genetics

Abstract

BackgroundOne of the purposes of life science researches is to elucidate the relationships between genotypes and phenotypes. However, it is often difficult to fill in information gaps in a target biological subsystem, and tremendous amounts of time and effort are required to reconstruct a subsystem's functions. Hence, we have proposed “bottom‐up genetics” as a novel methodology that enables high‐throughput determination of sets of genes required for the functioning of any biological subsystem [1, 2].MethodsBottom‐up genetics utilizes artificial cells, including a randomly introduced whole gene library, PURE system (reconstituted in vitro transcription and translation), and a reporter that fluoresces only when a particular function of a target biological subsystem is active. The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent cells and multiplex NGS analysis of genes contained in these cells. The importance of this methodology is that screening for the set of genes involved in a target biological subsystem and reconstituting the entire subsystem can be done simultaneously.Results & DiscussionWe tried to reconstruct β‐galactoside hydrolysis subsystem of Escherichia coli using bottom‐up genetics. We prepared an artificial cell library that contained the PURE system solution, 100 μM CMFDG (fluorescence reporter), and 5 nM E. coli ORF library. We isolated fluorescent and non‐fluorescent cells by FACS, and the genes included in them were determined by single‐cell NGS analysis. As a result, we succeeded in determining necessary and sufficient gene for β‐galactoside hydrolysis. This result indicated successful high‐throughput reconstruction of the β‐galactoside hydrolysis subsystem from the E. coli ORF library by bottom‐up genetics.Support or Funding InformationThis work was supported by JSPS KAKENHI (Grant No. 26830139).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.