Team:Glasgow/Project/Switch

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The Switch

The switch is the central part of the project, and the key to making our new system function. We constructed the recombinase-activated switch using a terminator, a reverse BBa_J23100 promoter and spacers needed because integrase works best with over 200 bases between att sites. We inserted the restriction sites, HinDIII and BamHI into the switch oligo. The size of the fragment changes when φC31 integrase is able to recombine the att sites making it clear on a gel whether the DNA has been recombined or not.
What else did we have to do?

• A reverse RFP made using PCR to attach the prefix and suffix backwards reversing the BBa_E1010 ORF.
• Used reverse RBS BBa_I742130 and made new more efficient reverse RBS using Ribosome Binding Calculator. (Insert results of how G-RBS is better)
• Used B0034 upstream of E0040 GFP (recreating BBa_J85201) and cloned downstream of the recombinase switch (Part)
• Cloned Reverse RBS-RFP construct upstream of the recombinase switch (PART) + BBa_J8201 construct
  This construct was cloned into a low copy number vector with a PSC101 origin. The nature of a switch requires a low copy number within a cell to avoid ‘leakage’ of the signal. This leakage is caused by some RFPs being transcribed when GFP should be or vice-versa. Therefore having fewer plasmids in a cell reduces the leakage problem.

The Integrase