Part:BBa_K847210

polA (DNA Polymerase I) DnaA-activated promoter

The polA promoter (polAP) is a DNA-replication dependent promoter.

Under optimal conditions, K12 E. coli will initiate DNA replication about every 10 minutes, given that a cell replicates about every 20 minutes and must account for a 40 minute chromosomal replication time (Grant 2011). Our results show oscillations at around a 10 minute period. Given that we grew our strains in fresh medium (LB) in a shaking 37C incubator, which constitutes near-optimal conditions, we can conclude that we expect ~10 minute period oscillations. From this we determine that the results suggest that our polAP promoter exhibits DNA-replication dependence, as shown in Table 2 and 3.

[[2: First bulk assay results for polAP]]

[[3: Second bulk assay results for polAP]]

Usage and Biology

DnaA is the central initiator of DNA replication in E. coli and other prokaryotic organisms, but importantly, it also functions as a transcription factor that can suppress or activate transcription of genes by binding to the DnaA box, a 9bp consensus sequence (Messer 1997). Since DnaA expression is dependent on the growth conditions of the cell (Chiaramello 1990), genes regulated by DnaA are transitively growth dependent. One such DnaA-dependent gene is polA, which codes for DNA Polymerase I, active in DNA replication (Quiñones 1997).

Sequence and Features

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]

Sources

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.