Concentration-Dependent CsrA Regulation of the uxuB Transcript Leads to Development of a Post-Transcriptional...

Post-transcriptional control systems offer new avenues for designing synthetic circuits that provide reduced burden and fewer synthetic regulatory components compared to transcriptionally based tools. Herein, we repurpose a newly identified post-transcriptional interaction between the uxuB mRNA transcript, specifically the 5' UTR + 100 nucleotides of coding sequence (100 nt CDS), and the E. coli Carbon Storage Regulatory A (CsrA) protein to design a biological post-transcriptional bandpass filter. In this work, we characterize the uxuB mRNA as a heterogeneous target of CsrA, where the protein can both activate and repress uxuB activity depending on its intracellular concentration. We leverage this interaction to implement a novel strategy of regulation within the 5' UTR of an mRNA. Specifically, we report a hierarchical binding strategy that may be leveraged by CsrA within uxuB to produce a dose-dependent response in regulatory outcomes. In our semisynthetic circuit, the uxuB 5' UTR + 100 nt CDS sequence is used as a scaffold that is fused to a gene of interest, which allows the circuit to transition between ON/OFF states based on the concentration range of free natively expressed CsrA. Notably, this system exerts regulation comparable to previously developed transcriptional bandpass filters while reducing the number of synthetic circuit components and can be used in concert with additional post-transcriptionally controlled circuits to achieve complex multi-signal control. We anticipate that future characterization of native regulatory RNA-protein systems will enable the development of more complex RNP-based circuits for synthetic biology applications.