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.

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

Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch

Figure5.The growing state of the reprogrammed bacteria with original Toggle-switch-Aptamer-cheZ Device(Red circle:the range of motion, Blue Triangle:the gradient of thepphylline)

Figure6.The fluorescent Photo(4X Objective) of the colony shown in Figure5 Left.

    According to the results above, by using this device we can not make the reprogrammed bacteria be divided into two different states effectively, and because of the bias to the green fluorescence and moving towards the high concentration of the theophylline, the bacteria trending to stay are very rare.

Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch

Figure7.

    Figure7 shows the fluctuation of expression of the modified Toggle Switch device, the number of bacteria with red fluorescent decreases from right to left.

Figure8.

    Figure8 shows four different growing states of the reprogrammed bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria(from the second tube（from right to left) shown in Figure7), and from left to right the concentration of the theophylline increases.

Figure9.The fluorescent Photo(4X Objective）of the colony1 in the fourth image of Figure8, and from left to right the concentration of the theophylline increases.

    Figure9 clearly display the dividing line between the reprogrammed bacteria at two different states, and the bacteria mainly express GFP indeed moving towards the high concentration of theophylline. This means the modified Toggle-switch-Aptamer-cheZ actually work as our design.

Acknowledgements

We want to thank IGEM Team of Perking University for providing toggle switches and advice on related experiments.

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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