Team:Melbourne/LinModel

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Contents

Full Diagram

Full Diagram

Components

Red light sensor

  • In the dark - EnvZ and OmpR are phosphorylated
  • With red light - EnvZ and OmpR are dephosphorylated
  • See Levskaya et al. Synthetic biology: engineering Escherichia coli to see ligh'. Nature (2005) vol. 438 (7067) pp. 441-2 for more detail


OmpF Promoter

  • Phosphorylated OmpR results in this promoter being repressed
  • Dephosphorylated OmpR results in this promoter being activated
  • See Rampersaud et al. The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation. J Biol Chem (1994) vol. 269 (17) pp. 12559-66 for more detail


Gene X/Y and Px/y)

  • Generic transcription factors that activate the corresponding promoter sequence
  • Candidate proteins/sequences being collected here


RiboLock and RiboKey

  • Riboswitch controls that prevent translation of a sequence with a ribolock unless the corresponding ribokey is also present
  • See Isaacs et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol (2004) vol. 22 (7) pp. 841-7 for more detail


Off X

  • As yet undetermined mechanism for switching off part 1
  • Could be riboswitch, transcription factor, or protein kinase/protease based
  • Must be self maintaining to keep part 1 off for entire cycle but further mechanisms (Off Y etc.) can be transient


GFP/RFP

  • Used as a reporter gene to determine what state the system is in (GFP = State 1, RFP = State 2)


Initial State

Initial State
  • Initially in the dark, therefore OmpR phosphorylated and OmpF repressed


First Pulse

First Pulse
  • Red light results in OmpR being dephosphorylated
  • Therefore transcription initiated at OmpF promoters
  • Gene Y transcript is locked off by RiboLock1 therefore no Gene Y is translated
  • Gene X is translated, which activates Px


Eventually...

First Steady State
  • Red light is switched off, therefore OmpF is repressed again
  • The activated Px produces more Gene X, the RiboKey1 and GFP
  • Therefore the system is in state 1 with state 2 prepared for activation on the next pulse of red light


Second Pulse

Second Pulse
  • Red light results in OmpR being dephosphorylated
  • Therefore transcription initiated at OmpF promoters
  • RiboKey1 is being produced, therefore RiboLock1 is opened and Gene Y can be translated
  • Gene Y activates Py which kickstarts the activation of part 2 and repression of part 1


Eventually...

Second Steady State
  • Red light is switched off, therefore OmpF is repressed again
  • The activated Py produces more Gene Y, RiboKey2, RFP and Off X
  • Off X repressed Px, switching off part 1
  • RiboKey 2 will open RiboLock 2 in part 3 (not shown)
  • RFP indicates that the system is in state 2 with state 3 (not shown) prepared for activation on the next pulse of red light


Extension

Each new part will require the following unique components

  • A Gene X/Px pairing
  • A RiboLock/RiboKey pairing
  • An Off component that will switch off the promoter for the previous part
                

Melbourne iGEM 2008

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