Module Integration

In order for us to achieve our goal of having a relay race between GFP and RFP teams,

various parts from the the three modules must be integrated together into a single working system. What follows is a description of the requirements for the integrated system.

Signal Plasmid

The signal plasmid will contain the following features:

• oriTR - to allow transfer during conjugation
• constitutively expressed trbK - to block back propagation of the signal, to reduce redundant transfer, and to prevent lethal zygosis
• constitutively expressed trbC - if lethal zygosis turn out to be a problem
• constitutively expressed GFP for the green team and RFP for the red team
• delay device
  • input: the delay is triggered by the presence of the helper plasmid
  • output: expression of the I-SceI homing endonuclease
• cut sites for the I-SceI homing endonuclease
• a low copy backbone to allow the endonuclease to destroy the signal plasmid

Helper Plasmid

This will consist of a modified R751 plasmid with the following features:

• trbK knockout to allow cells containing the helper plasmid to conjugate
• oriTR knockout to prevent transfer of the helper plasmid during conjugation
• trbC knockout to prevent lethal zygosis
• constitutively expressed trigger for the delay device

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