Part:BBa_K557002

Toggle-switch+Aptamer+CheZ

This part is the assembly of riboswitches followed by an important gene CheZ in keeping E.coli motile (BBa_K557001) finely tuned by small molecules and toggle switches (BBa_K228003) pushed on and off while memorizing current state. It would make it possible that bacterial colonies will automatically divide into two parts after certain time, during which riboswitches would act as the main power to drive two parts away from each other while toggle switches would play the role of giving birth to two 'different' kinds of bacteria in one colony.
With the help of BBa_K557003, it can produce more balanced colony division.
File:NO1.jpg
File:NO2.jpg

Model

Figure 1: Constructions to modulate the ratio of toggle switches. The above bar represents pSB3T5, a low to medium copy BioBrick standard vector, where LuxpR (BBa_R0062) controls the expression of cI repressor which would influence the ratio of toggle switch. (Images by USTC-IGEM with TinkerCell)

Experimental Data

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)

Acknowledgements

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.

Reference

1.Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 10
  Illegal AgeI site found at 122
  
• 1000
  COMPATIBLE WITH RFC[1000]