Regulatory
polAP

Part:BBa_K847210:Design

Designed by: Bryce Bajar, Jason Hu, Chris Jackson, Debha Amatya, Ben Geilich   Group: iGEM12_Stanford-Brown   (2012-07-24)

polA (DNA Polymerase I) DnaA-activated promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Potential cell-cycle dependent promoters in Escherichia coli and their sequences were identified from literature (Quiñones 1997) and isolated using Colony PCR. We developed our constructs for assay by ligating these PCR products in front of BBa_E0840, which consists of RBS BBa_B0030, reporter BBa_E0040 (mut3b GFP), and terminator BBa_B0015.

This construct functions in E. coli but may also work in prokaryotes with similar DnaA dynamics (e.g. Caulobacter).

Source

E. coli strain K12

References

Messer M., Weigel C.: DnaA initiator - also a transcription factor. Molecular Microbiology 1997, 24:1-6.

Chiaramello A.E., Zyskind, J.W.: Coupling of DNA replication to growth rate in Escherichia coli: A possible role for guanosine tetraphosphate. J. Bacteriology 1990, 172:2013-2019.

Quiñones A., Wandt G., Kleinstauber S., Messer W.: DnaA protein stimulates polA gene expression in Escherichia coli. Molecular Microbiology 1997, 23: 1193-1202.

Messer W.: The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication 2002, 26:355-374.

Lam A., St-Pierre F., Gong Y., Marshall J.D., Cranfill P.J., Baird M.A., McKeown M.R., Wiedenmann J., Davidson M.W., Schnitzer M., Tsien R.Y., Lin M.Z.: Improved dynamic range of genetically encoded FRET sensors with bright new green and red fluorescent proteins. Nature Methods 2012, 9:1005-1012.

Ferullo D.J., Cooper D.L., Moore H.R., Lovett S.T.: Cell cycle synchronization of E. coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods 2009, 48:8-13.

Grant M.A.A., Saggioro C., Ferrari U., Bassetti B., Sclavi B., Lagomarsino M.C.: DnaA and the timing of chromosome replication in Escherichia coli as a function of growth rate. BMC Systems Biology 2011, 5:201.