BYU iGEM/Notebook/28 January 2011
Circuit Design Brainstorming
We spent the day coming up with various mechanisms to express a desired gene in the presence of ROS and heat. Once expressed properly, we would like our marker gene of choice to be expressed constitutively. Following are four proposed mechanisms. The mechanism we have decided to try is the fourth mechanism, which we have named the "SuperFlp Mechanism." Below is a diagram.
1. Basic FLp Recombinase Mechanism: A DNA section that is transcribed normally in the presence of ROS, but there is nothing important downstream. When heated, the promoter inverts (5' to 3' as well as switching strands), leading to transcription in the opposite direction, which contains our desired marker.
2. IRES Mechanism: The promoter on the strand is ROS activated, but the mRNA lacks a Shine Dalgarno sequence. The SD sequence will be inserted by a heat-activated internal ribosomal entry site (IRES) in the proper position to result in translation of the marker protein.
3. Riboswitch Mechanism: The presence of ROS leads to translation of protein A. Protein A is required for translation of the desired mRNA coding for the marker. The mRNA starts with a heat-activated riboswitch flanked by LoxP sites and followed by the Cre protein. So, when activated by heat, the heat-sensitive riboswitch will be eliminated, allowing constituent translation. The Cre sequence is followed by a duplicate sequence for protein A.
4. SuperFlp Mechanism: (Method of choice) An ROS transcription factor is followed by a riboswitch and a Flp recombinase. In the presence of both ROS and heat, the recombinase is expressed, flipping non-local promoter to the desired position. The big question is whether the Flp recombinase's work will be undone by time or the same Flp recombinase. If the recombination is permanent, the E. Coli will continue to express the desired marker even when no longer in the presence of ROS or heat.