Composite

Part:BBa_M36604:Design

Designed by: Sibel Sayiner, Justo Caballero, and Shamik Mascharak   Group: Stanford BIOE44 - S11   (2012-12-05)

Heat-->PoPS (with postive feedback loop)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1108
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1108
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1108
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1108
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 810
    Illegal SapI.rc site found at 1159


Design Notes

It should be noted that E. coli has a built-in negative feedback to sigma-32, such that an abundance of sigma-32 turns off natural sigma-32 production. In regards to our sensor, this behavior leads to a sinusoidal relationship of expression (of sigma-32 and one's actuator further downstream, though this should eventually reach a steady-state. However, we found that creating a positive feedback loop in our construct increased the likelihood of mutations in the rpoH coding strand since a cell was unable to handle excess sigma-32 (created due to the leakiness of the promoter) when in a non-stressed environment. When this composite was first designed, the primary focus was on guaranteeing that significant sigma-32 would be produced in order to guarantee high expression of the selected actuator. This, however, is not a necessary precaution.

Source

htpG Heat Shock Promoter and Sequence for Sigma-32 Transcription Factor are from the E. coli genome. The bicistronic RBS is from the BIOFAB database. Find more information at the associated part page.

References

Excess Sigma-32 leading to possible mutations: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC211317/?page=5

Cyclic cell response to Sigma-32: http://www.researchgate.net/publication/20461625_The_activity_of_sigma_32_is_reduced_under_conditions_of_excess_heat_shock_protein_production_in_Escherichia_coli

More information on the Sigma-32 Promoter: Erickson, J. W., and C. A. Gross. "Identification of the Sigma E Subunit of Escherichia Coli RNA Polymerase: A Second Alternate Sigma Factor Involved in High-temperature Gene Expression." Genes & Development 3 (1989): 1462-471. National Center for Biotechnology Information. Web. 27 Oct. 2012.