Part:BBa_K1100144

J23104-ALeaderT

As shown in Figure 1, the 75nt RNA sequence of Aleader contains two SD sequences (ribosome binding sites) and an anti-SD sequence (CUUC) which can complementarily pair with either of the SD sequences. In the absence of aminoglycosides, anti-SD pairs with SD2. The binding of ribosomes to SD1 triggers the translation of a small peptide which stops at the stop codon ahead of SD2, and therefore inhibits the translation of the desired gene after SD2. When aminoglycosides (kanamycin for example) exists, it will induce a structural change of Aleader. The anti-SD sequence base-pairs with SD1, consequently unmasking SD2 for ribosomal binding, which results in the translation of the following gene.

Figure1. Proposed Model for the Induction of Aminoglycoside Resistance: Aminoglycoside binding to the leader RNA induces a change in the leader RNA structure(Jia X et al, 2013.).

Many riboswitches have lower dynamic range and higher leakage rate compared with protein regulators. To reduce the basal level and enhance the dynamic range, while having no effect on the protein product, we designed a novel riboswitch called ALeaderT, following the principle of three-phase switch design.

We added a GGGS linker (dock III), a common neutral peptide linker, after the SD2 and ATG. Impressively, by RNA 2D-prediction, we found that dock III will capture antiSD2 sequence to form a stable stem loop, with characteristics of terminator, though neutral as an amino peptide. We suppose that the ALeaderT will have three different steady-state conformations, A “Translation off Termination off”, B “Translation on Termination off”, and C “Translation on Termination on”(Figure 2).

Figure 3.Tri-Phase Model of AleaderT.

We design a series of experiments to test the function of ALeaderT with the reporter mRFP1 and a promoter library(Figure 3). It showed a very low minimal level and high dynamic range, consistent with the previous prediction. Moreover, it changed the hill function working curve into a square-like curve. Surprisingly, the ALeaderT also showed virtues of marvelous low noise in the system and minimal difference under different regulation. Therefore it will have its talent on the precisely regulation of RNA and protein expression.

Figure 3. The Promoter Library for Quantitative Estimation of ALeaderT.

We tested the ALeaderT’s function on translation control with the reporter mRFP1 and a promoter library measured by fluorescent.

Figure 4. The dose-response curve of ALeaderT with a promoter library, measured by mRFP fluorescence test.

It showed a very low basal level and high dynamic range, consistent with the modeling prediction(Figure 5,6). Marvelously, the ALeaderT is also much more standardized and robust than the wild type ALeader(Figure 5). In other words, with different promoters, AleaderT’s function varies insignificantly. Therefore it is talented on the precisely regulation of RNA and protein expression.

Figure 5. ALeaderT has lower basal level than ALeader. The highest levels of them are not significantly different, though ALeaderT have a little higher highest level. Moreover, the robustness of ALeaderT is better than the wild type ALeader. T_Highest: highest level of ALeaderT, T_Basal: basal level of ALeaderT, A_Highest: highest level of ALeader(wt), A_Basal: basal level of ALeader(wt)

Figure 6. The AleaderT shows a higher dynamic range than the wild type ALeader.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]