Aptamer-CheZ: theophylline-sensitive synthetic riboswitch+phosphatase which dephosphorylates CheY-P
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
Chemotaxis has always been a powerful tool in organizing bacteria’s movement and it’s always an important ability to reprogram bacteria to follow entirely new chemical signals. Riboswitches, which have been a hot issue of synthetic biology recently, can be used to guide E.Coli toward and precisely localized to a completely new chemical signal. We take advantage of them in our system to act as the main power to drive two parts of a bacterial colony away from each other.
Figure 1: Model for how the theophylline-sensitive synthetic riboswitch controls the translation of the CheZ protein. In the absence of theophylline (left),the mRNA adopts a conformation in which the ribosome binding site is paired and translation of CheZ is inhibited. In the absence of CheZ, the protein CheY-P remains phosphorylated and the cells tumble in place. In the presence of theophylline (shown in red), the mRNA can adopt a conformation in which the ribosome binding site is exposed and CheZ is expressed, thus allowing the cells to run and tumble. (Images from: Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811)
Dose-Dependent Migration Ability of Aptamer-cheZ Device
To test the migration ability, selective media (tryptone broth with different ratio of agar(0.25%, 0.3%, 0.4%), 50μg/mL ampicllin, and various concentrations of theophylline(0mM, 0.25mM, 0.5mM, 0.5mM, 0.75mM, 1mM)) was prepared in Petri dishes(85mm dia). Diluted cell suspensions from mid-log-phase cultures were applied to the center of the plates, which were dried at room temperature at for 15 min, and incubated at 37°C for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth.
Figure2.((a)0.25%agar, (b)0.3%agar, (c)0.4%agar)
The results(Figure2 & 3.) show that the Aptamer-cheZ Device is functional, and the 0.3%agar is the best ratio for the migration experiments.'
Figure3. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Thephylline, Right:0.3%agar with 0.25mM Thephylline)
Here we want really want to thank the Professor J. S. Parkinson for providing ΔcheZ strain RP1616 and the corresponding wild type RP437 and Professor J. P. Gallivan for directing our construction of riboswitches.
1.Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811