## 3 Results
### Characterization of Switchable Terminators  
To benchmark the regulatory performance of designed SWT, we chose reference constructs among STAR and THS libraries. For STAR, STAR AD1 (STAR target WT) and the best STAR in the computationally designed library (STAR target 1) were selected [30,33]. For THS, three switch-trigger pairs were chosen based on their high fold-change values reported: ACTS_TypeII_N1 (THS A), ACTS_Typell_N7 (THS B), and ACTS_TypeII_N3 (THS C) [36]. The SWT candidates were tested in _E. coli_ BL21 (DE3) strain with the SWT and trigger RNAs expressed from separate medium and high copy plasmids, respectively (Figure 3a). Expression of trigger RNA was induced using IPTG, which initiated the production of SWT trigger RNAs through T7 RNA polymerase. GFP was used to characterize switch output performance via flow cytometry. The BBa K86400 SWT showed a fold change of about 10 and some noticeable leakage levels comparable to the STAR target WT (Figure 3b). On the other hand, the T500 SWT exhibited a nearly 900-fold change with a very low leakage level, providing similar performance to the best STAR reported (STAR target 1). To test whether the ON/OFF regulation of the SWT is due to the transcription regulation, we performed quantitative reverse-transcription PCR and determined the level of GFP mRNA in the presence and absence of trigger RNA (Figure 3c). As a control, THS C was used, which is regulated at the translational level rather than the transcriptional level [36]. We observed that the Ct values normalized with the conventional reference gene (16S rRNA) corresponded to high and low amounts of GFP mRNA for ON and OFF state of the SWT as expected. Of note, the T500 SWT showed the minimum amount of RNA in the OFF state when compared to BBa K864600 and STAR target WT while maintaining a  
Figure 3: In Vivo Characterization of SWT Candidates. (**a**) Transcription terminators T500 and BBa K864600 were engineered to be responsive to trigger RNA, and these were characterized in vivo. STAR target WT and STAR target 1 were included to benchmark performance. The GFP fluorescence of SWT candidates was measured in the presence of trigger RNA or decoy RNA. Fluorescence levels were expressed on a logarithmic scale. (**b**) ON/OFF ratios of SWT candidates and STARs were calculated. T500 SWT showed a high-fold change value of 879-fold with low OFF-state fluorescence. Relative errors for the GFP fluorescence are from the s.d. of three biological replicates. (**c**) Quantitative reverse-transcription PCR results targeting GFP mRNA for SWT candidates and STAR target WT. THS C was used as a control. Ct values were normalized by subtracting the Ct values of 16S rRNA for each sample, and the number above each column indicates the gap between Ct values in the presence of trigger RNA and decoy RNA. large Ct value difference of 7.34 cycles for the ON and OFF states. For RT-qPCR results on the reference gene, 165 rRNA, we observed that there was a very small difference in raw Ct values (typically less than 0.2 cycles) in the presence and absence of cognate trigger RNAs (Table S9). This suggests that the Ct value differences in Figure 3c could reflect the changes in the absolute amount of target GFP mRNA. For T500 SWT, this translates to about a 160-fold difference in the amount of target GFP mRNA, somewhat less than the fold change in GFP fluorescence observed. Thus, the T500 SWT represents a new transcriptional regulator with excellent dynamic features, and therefore, we further characterize and expand T500 SWT through subsequent experiments. Next, we aimed to investigate the design flexibility of T500 SWT by modifying the critical stem-loop sequence and asked whether it maintains its characteristics as an SWT (Figure 4a). We changed 3 base pairs at the bottom of the hairpin, which is considered critical to the terminator function [43].