Project

Part:BBa_M36778:Design

Designed by: Evan Clark   Group: Stanford BIOE44 - S11   (2011-05-03)

DNA damage sensor with Gemini Reporter (weak LexA Generator)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 782
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 782
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 782
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 782
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 782
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1759


Design Notes

Our device utilizes the machinery of the SOS response pathway to detect DNA damage. LexA acts as a repressor for the genes in the SOS response pathway by binding to their promoters and preventing transcription. In the presence of DNA damage, another protein, RecA, binds to ssDNA. The RecA-ssDNA complex interacts with LexA, causing it to cleave off the promoter, and allowing transcription of the SOS genes to go forward. In a general sense, our device attaches a SOS gene promoter to the Gemini reporter so that Gemini will be expressed whenever the SOS response is activated (whenever DNA damage is present). Specifically, we chose the LexA promoter as the promoter to use, because LexA has the highest promoter activity of the genes in the SOS response pathway. LexA is self-regulatory.

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 LexA generator upstream of the sensor sequence. This generator consists of a medium strength constitutive promoter and a weak strength 5'UTR attached to the LexA gene and a terminator. The strength of the promoter and 5'UTR can be varied in an attempt to do PoPS level matching; we have designed another part, BBa_M36054 with the same promoter and a medium strength 5'UTR for this reason.

Source

Parts registry, E. coli genome (LexA gene and promoter)

References

Analysis of Escherichia coli RecA Interactions with LexA, CI, and UmuD by Site-Directed Mutagenesis of recA JULIE A. MUSTARD1 and JOHN W. LITTLE

Control of recA Gene RNA in E. coli: Regulatory and Signal Genes Ann McPartland, Linda Green and Harrison Echo

DNA binding properties of the LexA repressor M Schnarr, P Oertel-Buchheit, M Kazmaier, M Granger-Schnarr

Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria Nir Friedman1, Shuki Vardi1[, Michal Ronen, Uri Alon, Joel Stavans

The bacterial LexA transcriptional repressor M. Butalaa, D. Zgur-Bertokb and S. J. W. Busbya

Assigning numbers to the arrows: Parameterizing a gene regulation network by using accurate expression kinetics Michal Ronen, Revital Rosenberg, Boris I. Shraiman, and Uri Alon

Structure of the LexA–DNA complex and implications for SOS box measurement Adrianna P. P. Zhang, Ying Z. Pigli & Phoebe A. Rice