Fig. 4: Developing a Csr-regulated NOT Gate (cNOT) and deploying in tandem with cBUFFER to engineer a bi-directional regulator.

A Native regulatory interaction of the of the ymdA mRNA transcript with the Csr Network. In the presence of CsrA, the mRNA is bound by CsrA that activates translation. When CsrB is transcribed and sequesters CsrA, the hairpin on the CsrA, the hairpin on the ymdA 5’ UTR forms and prevents the ribosome from binding. B Genetic circuit diagram of the cNOT system - CsrA activates translation of the target gene through the engineered ymdA 5’ UTR. When CsrB is transcribed, it sequesters CsrA causing translation de-activation of target. C Time course of the cNOT fluorescence for induced and uninduced cultures. D Diagram of the preliminary bi-directional regulator – CsrA regulates mcherry via cBUFFER and gfpmut3 via cNOT. E Time course of relative fluorescence for GFP and mCherry in the bi-directional using wild type CsrB. F Fold change of each fluorescent target two hours post-induction. G Genetic circuit diagram of bi-directional regulatory on a single operon. H Time course single operon bi-directional regulator. I Relative fold change of GFP and mCherry signal from the single synthetic operon bi-directional regulator two hours post-induction. Samples were grown in biological triplicate, and data presented are the mean values +/- the standard deviation, represented as the error bars.