IGEM:IMPERIAL/2006/project/Oscillator/project browser/Prey Construct/Design: Difference between revisions

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*The prey part: [http://parts.mit.edu/registry/index.php/Part:BBa_J37015 J37015 @ MIT Parts Registry]
*The prey part: [http://parts.mit.edu/registry/index.php/Part:BBa_J37015 J37015 @ MIT Parts Registry]


[[Image:J37015 logo.png]]
[[Image:J37015page.JPG]]


==Design Choices==
==Design Choices==


*Design allows for an exponential increase of AHL via a positive feedback loop
*Design must fulfil the [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Prey_Construct specifications for the prey construct].
**Interaction between LuxR, AHL and promoter controlling expression of LuxI
*Part design will be based on Quorum-sensing/quenching and the availability of BioBricks.
::*Research into the registry directed us to use AHL as our prey molecule.
*Design allows for an exponential increase of AHL (to fulfil the [[IGEM:IMPERIAL/2006/project/Oscillator/Theoretical_Analyses/2D_Model1|Lotka-Volterra model]]) via a positive feedback loop.
*The prey part can be decoupled into a prey sensing part and a prey producing part.
*Initiation of positive feedback loop depends on leaky expression of the tetR promoter which is a constitutive promoter.
** This leaky expression produces LuxR which can form a dimer with AHL already withing the cell.
** LuxR, AHL and promoter control the expression of LuxI which produces AHL.
*Design allows for assesment of status of positive feedback loop via:
*Design allows for assesment of status of positive feedback loop via:
**GFP Production - Linked to LuxI expression
**GFP Production - Linked to LuxI expression
**AHL Production - Can be indirectly assessed via T9002 assay
**AHL Production - Can be indirectly assessed according to [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols/J37015 J37015 protocol].
 




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! INPUTS !! biological component !! comments
! INPUTS !! biological component !! comments
|-
|-
|AHL||LuxR + pLuxR||These components sense input and activate output
|AHL||LuxR & pLuxR|| To initiate the positive feedback loop, LuxR is produced via leaky expression of the tetR promoter.
|- style="background:orange"
|- style="background:orange"
! OUTPUTS !! biological component !! comments
! OUTPUTS !! biological component !! comments
|-
|-
|AHL||LuxI||LuxI expression causes enzymatic generation of AHL  
|AHL||LuxI||LuxR, AHL and pLuxR form a complex, activating production of LuxI expression which causes enzymatic generation of AHL  
|}
|}
==Open Issues==
*We need to consider methods of controlling the positive feedback loop while cells are growing to ensure AHL concentration stays at zero. Possible suggestions are:
::*Periodic dilution of culture to allow degradation of LuxI
::*Riboswitch
::*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/popsblocker Pops blocker]
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Latest revision as of 09:40, 30 October 2006

Super Parts Molecular Prey-Predator Oscillator
Actual Part
Sub Parts Test Sensing Prey Construct <bbpart>C0261</bbpart> <bbpart>I13401</bbpart>


Registry

Design Choices

  • Research into the registry directed us to use AHL as our prey molecule.
  • Design allows for an exponential increase of AHL (to fulfil the Lotka-Volterra model) via a positive feedback loop.
  • The prey part can be decoupled into a prey sensing part and a prey producing part.
  • Initiation of positive feedback loop depends on leaky expression of the tetR promoter which is a constitutive promoter.
    • This leaky expression produces LuxR which can form a dimer with AHL already withing the cell.
    • LuxR, AHL and promoter control the expression of LuxI which produces AHL.
  • Design allows for assesment of status of positive feedback loop via:
    • GFP Production - Linked to LuxI expression
    • AHL Production - Can be indirectly assessed according to J37015 protocol.


INPUTS biological component comments
AHL LuxR & pLuxR To initiate the positive feedback loop, LuxR is produced via leaky expression of the tetR promoter.
OUTPUTS biological component comments
AHL LuxI LuxR, AHL and pLuxR form a complex, activating production of LuxI expression which causes enzymatic generation of AHL

Open Issues

  • We need to consider methods of controlling the positive feedback loop while cells are growing to ensure AHL concentration stays at zero. Possible suggestions are:
  • Periodic dilution of culture to allow degradation of LuxI
  • Riboswitch
  • Pops blocker

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