Designed by: Huachun Liu   Group: iGEM12_ZJU-China   (2012-08-09)

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).

Secondary structure version 2.jpg Tertiary structure version 2.jpg

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).

Control mechanism of the theophylline aptamer.jpg

Fig 2 The control mechanism of the theophylline aptamer.

Three versions of clovers.jpg

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.

Test of fluorescence.jpg

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

Fig 5 confocal image.jpg

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
  • 12
  • 21
  • 23
  • 25
  • 1000

Functional Parameters: Austin_UTexas

BBa_K738002 parameters

Burden Imposed by this Part:

Burden Value: -0.4 ± 1.8%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

n/aTheophyline riboswitch regulated RNA Scaffold