Sensor 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. We expect to have these results in time for the jamboree.

World Championship Update

In order to test to make sure our variations of RBS+cI and RBS+Cro were working as expected, we designed four test circuits. Our ultimate goal was to have GFP under the control of the Prm promoter of Or and mCherry under the control of the Pr promoter of Or. With this design, we would be able to tell the effect each of the three cI repressors and Cro had on the two different promoters. We put the cI repressors and Cro under the control of the arabinose inducible promoter pBAD, so we could control their expression. Once induced, the cI repressors or Cro would be transcribed and depending on how they bound to the binding sites of the Or operating region, we would be able to see expression or lack of expression of the two fluorescent proteins. As of the wiki freeze, we have successfully constructed 4 plasmids containing all of the components of our test circuits other than B0034+GFP. We are currently in the process of attaching the B0034+GFP to the circuit as well as collecting data on the effect that the three cI repressors and Cro have on the Pr promoter.