Team:NTU-Singapore/Project/Prototype

Our Prototype

In the Research Proposal, we identified the gene sequences of interest that will allow us to build our prototype system.

Now we can start to design our system from the ground up, starting with arranging parts into devices.

We can also begin to construct a top-down modelling schema to simulate our system! This schema can be further refined by studying our devices on their own.

Prototype System Design

Recall the construct layout for the ideal system earlier. Our prototype will utilize a similar layout.

We will now elaborate on the rationale of parts placement in each device.

Sensing device

• Arguably the most important gene sequence, we have decided to use the NO-sensitive promoter region of the NorV/W gene sequence endogeneous in E.Coli.
  • This NO-sensitive promoter (we will refer to it as pNO henceforth) will be the active NO-sensor for our system.
    • pNO is ON in high [NO] conditions & OFF in low [NO] conditions.

• The pNO will be placed upstream of a TetR gene sequence. TetR is used to repress the other devices.
  • The rationale here is that in healthy [NO] condition, pNO is activated and will facilitate expression of TetR.
  • In low [NO] conditions, pNO will be deactivated and the transcription of TetR gene sequence will stop. Hence repression of other devices will stop.

Degradation device

• As mentioned above, the pTet promoter will serve as the controller for this device.
  • In high [TetR] conditions, implying high [NO] conditions, Degradation device is OFF. In low [TetR], implying low [NO] conditions, this device will be ON.

• LuxI gene sequence expresses LuxI protein.
  • LuxI protein catalyzes conversion of SAM protein to AHL.
  • This conversion has significance in the Dilation device.

• CHE gene sequence will express Cholesterol Esterase enzyme.
  • This enzyme will breakdown the cholesteryl esters in plaque.

Imaging device

• Again, pTet regulation will control the activation/repression of this device.
  • System is only ON at low [NO] and OFF at high [NO].

• The Heme-Oxygenase 1 gene sequence (HO-1) and Infrared Protein gene sequence (IFP) are to be considered coupled.
  • HO-1 expression will facilitate the conversion of heme to biliverdin.
  • IFP, a protein discovered by Tsien lab, requires bilverdin to be stable and fluoresce in the infra-red spectrum.

(Proposed) Dilation device

The Dilation device is special because it features a "delay" mechanism endemic to E.Coli. This delay mechanism will be replaced with a mammalian counterpart if designing the actual mammalian biological system.

• The Dilation device features constitutive transcription of LuxR gene sequence.
  • [LuxR] will repress the activation of pLuxR further downstream.

The following is our E.Coli delay mechanism.

• pLuxR is activated when LuxR is removed by selective binding to AHL.
  • This is where the LuxI from the Degradation device comes in.
    • LuxI converts SAM protein to AHL. AHL binds to LuxR. We expect this series of binding comes with a time lag.
  • Recall that Degradation device is only ON at low [NO] conditions.
    • Hence pLuxR is only activated at low [NO] conditions, and only after a lag period.

• Activated pLuxR allows for transcription of Nitric Oxide Synthase [NOS].
  • NOS will then regenerate [NO] at plaque site.
  • Regeneration of [NO] to normal levels can be expected to trigger the sense construct thus shutting down every other construct.

System Overview

The following is the overall genetic circuitry for our prototype system.

As we detailed the part placement in each device, this circuit is complete. Our aim now is to translate this design into actual construct-Biobricks!

System Modelling

But first, let's try to model our system using blackboxes. We already have designed a coherent system complete with devices with specific objectives and parts with well-defined gene sequences. To analyze our system theoretically & mathematically, we must have a logical circuit.

The following is the Simulink circuit we have designed for our system. Please click on it for a larger view.

The advantage of laying out the system as a combination of Simulink sub-systems/devices is that it allows us to visualise each step of the circuit as a blackbox, with only the input and output visible. This simplifies the study of the system infinitely.

From the Simulink layout, we can understand that

• Sensing device activation/deactivation depends on the net [NO] from external surrounding and the generated contribution.

• Both the Imaging device & Degradation device depend exclusively on [TetR] for activation/efficiency.
  • Biliverdin generation depends on [HO-1].
  • Infrared signal depends on IFP - HO-1 binding efficiency.

• Delay mechanism depends on conversion efficiency of SAM to AHL by LuxI.

• Activation/efficiency of the Dilation device depends on binding efficiency of LuxI & AHL.

• And finally, we realise that regeneration of [NO] depends on [NOS].

Knowing all these, we can identify bottlenecks, and account for any deviations in expected curves in actual construct characterization. Now we can start looking at each device one by one. From here on, our system descriptions will be a bit more technical. Bear with us!

Proceed here to see our comprehensive section on Sensing Device.

Click here to see details about the Degradation Device.

And finally, you can see our section on Imaging Device here.

Literature / References

Please proceed here to view our full list of references.