Part:BBa_M36778
DNA damage sensor with Gemini Reporter (weak LexA Generator)
This device is a sensor to detect DNA damage in the form of double-strand breaks or regions of single-stranded DNA arising from stalled replication forks. The device indicates the presence of such damage using the Gemini reporter, and utilizes the machinery of the SOS response pathway to detect DNA damage.
The SOS response pathway is a DNA repair pathway in E. coli and other bacteria. The ~40 genes of the SOS repair pathway are regulated by LexA, a repressor that binds to their promoters and prevents transcription. In the presence of single-stranded DNA (from DNA damage), another protein, RecA, binds to the ss-DNA. The recA-ssDNA complex interacts with the LexA bound to the SOS gene promoters, causing it to cleave off and allowing transcription to go forward.
Our device utilizes this fact to create a DNA damage sensor by attaching the Gemini reporter behind a SOS gene promoter. In the presence of DNA damage, the RecA-ssDNA complex will cause LexA to cleave off the promoter, allowing transcription of Gemini to go forward. LexA is a self-regulatory SOS gene, and has one of the highest promoter activities of the SOS genes, so we chose the LexA promoter as the promoter to drive our device.
We were concerned that the native concentrations of LexA in the cell would not be high enough to support our added sensor infrastructure, so this device includes a constitutive LexA generator upstream of the sensor sequence.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 782
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 782
Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 782
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 782
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 782
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1759
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