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From 2011.igem.org

RecA Project Sensor Project Reporter Project

The Sensor

The design of our sensor was based on the lambda phage lysogenic vs lytic switch. Optimally, the system would be activated in the presence of DNA damage due to radiation. For that reason, we utilized the lambda phage switch, which transitions from the lysogenic cycle to the lytic cycle when DNA damage is detected. The genetic circuit we designed focuses on the DNA damage-sensitive lambda phage lytic-lysogenic cycle switch followed by a rapid response reporter similar to the immobilized fusion enzyme system pioneered by the Imperial College of London 2010 iGEM team. An initial design of our sensor circuit is illustrated below.

Under normal conditions, PRM is active, which transcribes the lambda repressor, cI + RBS. The lambda repressor binds to OR1 and OR2, repressing PR, which in turn inhibits the transcribtion of Cro and TEV.

Once DNA damaged occurs, the ssDNA binds to RecA. RecA becomes activated and cleaves the cI repressor, allowing for PR to turn on. Cro then binds to OR3, which represses PRM and inhibits the transcription of the cI repressor. Now that PR is active, the transcription of the TEV protease occurs, which is used in the reporter structure of the circuit.

Accomplishments:

We have successfully completed the construction of three different strength RBSs attached to the cI repressor as well as the construction of the OR (operating region for the Lambda switch) with an RBS + Cro, an RBS + Tev, and a terminator. We are currently working on attaching each RBS + cI repressor onto our other construct previously stated.

Future Plans:

Once our final construct is complete, the next step will be to add our RecA mutant under the control of a constitutive promoter, followed by the addition of RFP and a terminator, so that it may be tested. This construct will be tested by using UV light for varying lengths of time, and subsequently measureing the fluorescence output. This test should provide data as to the range of DNA damage that will be detected for each variation of RBS + cI we constructed.