Difference between revisions of "Part:BBa K847210"

Revision as of 21:58, 3 October 2012

polA (DNA Polymerase I) DnaA-activated promoter

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

K847210 displayed fluorescence when transformed into NEB-5alpha competent cells. However, when ligated in front of E0840, the signal was too weak for the camera to recognize. The polA promoter was therefore digested with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness (Lam 2012). This sufficiently bright polAP-Clover construct is displayed in Figure 1.

Table 1: microscopy shows polA promoter activity. C: GFP filter. D: phase

To characterize the cell-cycle dependency of K847210, performed a bulk assay to compare growth and fluorescence. We used serine hydroxamate (SHX) to synchronize cell cycle (Ferullo 2009), washed the cells in LB, then incubated cells at 37C. These samples were then measured regularly for OD600 and fluorescence.

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.

Table 2: First bulk assay results

Table 3: Second bulk assay results

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

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.