Fluorescence-Activated Cell Sorting Enables Rapid Screening of RNA Polymerase–Promoter Interaction in E. coli

Abstract

RNA polymerases are essential in the enzymatic synthesis of RNA via in vitro transcription. The resulting mRNA transcripts have been investigated more and more as therapeutics in clinical studies. Therefore, it is important that these enzymes possess a high specificity. Despite their utility, many commercially available RNA polymerases are limited by their tendency to generate abortive transcripts and undesired byproducts. To ensure high specificity, optimal pairing between the RNA polymerase and its cognate promoter is vital. However, current promoter characterization techniques remain laborious and time-consuming, thereby limiting efficient and rapid RNA-polymerase applications. In this work, we describe a high-throughput strategy for rapid promoter identification using a two-plasmid screening platform in E. coli. Cell populations are analyzed and sorted via fluorescence-activated cell sorting (FACS), followed by sequence verification. Functionality of the screening platform was validated via cell sorting, based on high fluorescence, of a mixed population containing T7 RNA polymerase paired with three different promoters. Moreover, the screening system was evaluated by using two recently identified RNA polymerases in combination with their respective cognate promoters. This proof-of-concept facilitates the identification of both RNA polymerase and promoter. Further, it substantially accelerates promoter characterization, thereby supporting improvements regarding mRNA manufacturing workflows.