## 3 Results
### Multilevel Regulatory System and 2-Input AND-Gate Implementation  
Building on the success of T500 SWT as a transcriptional regulator, we designed a multilevel regulator that simultaneously controls transcription and translation by concatenating the T500 SWT with the well-characterized translational regulator THS. The SWT should be turned on by the cognate SWT trigger RNA to allow transcription of the downstream THS connected to the target gene (Figure 5a). In the absence of the THS trigger RNA, the RBS remains enclosed within the stem-loop of THS, preventing the ribosome access and translation of the target gene [31]. If cognate THS trigger RNA is also present, the hairpin structure of THS could be unwound by the trigger RNA, exposing RBS and start codon to activate translation of the downstream gene (Figure 5b). Note that this multilevel regulatory system is essentially functioning as a 2-input AND gate where the target gene is expressed only when both SWT trigger RNA and THS trigger RNA are present (Figure 5c). Since target gene expression is regulated both at transcription and translation levels, tighter expression control may be achieved when compared to regulation at a single stage.  
Figure 5: Implementation of 2-input AND Gates via Multilevel Regulatory System. (**a**,**b**) Design schematics of a multilevel regulatory system that concatenates SWT and THS in the same expression cassette. Transcription is controlled by SWT trigger RNA (**a**), and the subsequent translation of the downstream gene is controlled by THS trigger RNA (**b**). Thus, the output gene expression can occur only in the presence of both SWT and THS trigger RNAs. (**c**) Schematics of the 2-input AND gate formed by the multilevel regulator. (**d**–**f**) The truth table for the AND computations: T500 SWT + THS A (**d**), T500 SWT + THS B (**e**), and an analogous construct previously reported [38] (**f**). The multilevel regulators that combine T500 SWT and THS showed very low leakage levels for (0, 1) and (1, 0) input combinations in contrast to the previous regulator shown in (**f**). (**g**) The OFF states were compared for multilevel regulatory circuits and the constituent single THS. The negative control contained an empty pCOALDuet plasmid. Relative errors for the GFP fluorescence are from the s.d. of three biological replicates.  
We created multilevel regulatory systems by connecting the T500 SWT with THS A (T500 A) and THS B (T500 B) from the second generation THS library [36]. For both multilevel regulators, the GFP expression patterns were consistent with the 2-input logical AND gate with SWT and THS triggers as inputs (Figure 5d,e). The multilevel regulators demonstrated very low GFP expression in all three logical FALSE conditions and strong GFP expression in the sole logical TRUE condition. To benchmark the performance of our multilevel regulators, we also tested a previously reported 2-input AND gate connecting the STAR target RNA and the first generation THS in the same transcript (Figure 5f) [38]. Notably, the leakage levels of our designs were lower than the OFF state of [PERSON]'s AND-gate (Figures S4 and S5). Additionally, the leakage level of multilevel regulators was much lower than those of constituent THS (Figure 5g), suggesting the improved leakage control and logic capability of SWT and THS combinations.