Figure 7. Application of a translational regulator to construct complex genetic circuits. The following three examples use the crRNA-taRNA, a riboregulator that controls translation initiation (Fig. 5A, Isaacs et al.,73 2004), to engineer different synthetic systems. (A) An adaptor that converts regulators from translational to transcriptional. By fusing an adaptor to a translational regulator, the translational regulator becomes transcriptional regulator. The adaptor consists of a Ribosomal Binding Site (RBS), an Open Reading Frame (ORF) of a short leader peptide (tnaC), and a transcription termination region. By fusing this element to the translational regulator (e.g., crRNA-taRNA), translation of the adaptor is controllable (not shown). In the absence of the adaptor, regulation of GFP is at the translation initiation level by RBS sequestration. In contrast, by merging the adaptor, regulation of GFP is at the transcriptional elongation level; in this case, when the upstream translational regulator turns off synthesis of the adaptor, the transcription terminator in this element is exposed and arrests transcription of GFP (at the mRNA level). Likewise, when the translational regulator turns on synthesis of the adaptor, translation of GFP is activated at the protein level.1 (B) Riboregulated Transcription Cascade (RTC) with the ability to count. By arranging 3 genes controlled by the translational regulator (crRNA-taRNA) into a transcriptional cascade a genetic circuit is built with the ability to count. When PBAD promoter is not induced, the crRNA element present in each gene blocks translation of the T7 and T3 RNA Polymerase (RNAP), and GFP. In contrast, when PBAD is induced with arabinose, the level of fluorescence generated correlates with the number of pulses of arabinose fed into the system.78 (C) Switchboard for multi-sensing and metabolic pathway control. A series of rationally designed orthogonal variants of the crRNA-taRNA system (cr1, cr2…crn; taRNA1, taRNA2…taRNAn) are fused to multiple gene reporters (Reporter 1, Reporter 2…Reporter n) for simultaneous and independent regulation. The complex genetic circuit is arranged in parallel (each pair of crRNA-taRNA variants is under the control of a different promoter) to be able to sense multiple inputs and convert them into a measurable output (e.g., enzymatic and/or fluorescence reads).79