Part:BBa_K738002

Theophyline riboswitch regulated RNA Scaffold (clover vision 2)

RNA scaffold D0 (BBa_K738000) was constructed from a single RNA module d0, which folded into a duplex with PP7 and MS2 aptamer domains that bind PP7 and MS2 fusion proteins. A designed theophylline aptamer was added on the original scaffold D0 in order to produce an interaction with MS2 aptamer in the absence of theophylline, thus disturbing the bind between MS2 aptamer and corresponding protein (Fig 1).

Fig 1 Secondary structure (left) and tertiary structure (right) of K738002

However, when theophylline is added, the fold of the loop is changed and thus the interaction will disappear, leading to the binding between MS2 aptamer and corresponding protein (Fig 2).

Fig 2 The control mechanism of the theophylline aptamer.

Fig 3 Three versions of clovers. Version 2 has adjacent MS2 and theophylline aptamer. It has an interaction between the loop of theophylline aptamer and the stem of MS2 aptamer.

RNA riboscaffold clover 2 can be regulated and controlled through conformational change by theophylline.

---

When the concentration of Theophylline is in the range from 0mM to 0.5mM, the concentration of Theophylline and the resulting fluorescence intensity are directly proportional (Fig 4). Theophylline concentration beyond certain extent will be hazardous to cells and how it affects cells depends on strain type.

Fig 4 Tests of fluorescence/ OD change over theophylline concentration. There’s evident positive correlation fluorescence intensity between concentration of theophylline added.

Fig 5 Different RNA scaffold’s effect on split GFP showing by fluorescence microscopy. The BL21*DE3 of the E. coli were transformed with pCJDFA+pCJDFB, pCJDFA+pCJDFB + pCJDD0, and pCJDFA+pCJDFB + pZCCOV 2 (0.5 mM theophylline adding). As expected, strains without RNA scaffold did not fluoresce. Upon the existence of RNA scaffold, many of the cells emitted fluorescence indicating a substantial amount of split GFP combination is permitted because of the function of RNA scaffold. The brightfield images in the right column depict all bacterial cells. The GFP images in the left column depict bacterial cells which emitted fluorescence.

Usage and Biology

This scaffold, by theophylline management, could have a variety of functions, more than accelerate the reaction, but whether to accelerate or not, the degree of acceleration and even reduce the reaction rate.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]