Difference between revisions of "Part:BBa K847211"

Revision as of 22:11, 3 October 2012

nrd operon promoter

The nrd promoter (nrdP) is a cell-division dependent promoter.

We first show that the part can promote fluorescence. We ligated K847211 in front of BBa_E0840 and observed fluorescence under microscopy as indicated by Figure 1.

To assay this characteristic, we used a bulk assay in which we use serine hydroxamate (SHX) to synchronize the cell cycles of a sample (Ferullo 2009), wash the cells of SHX, then incubate the sample at 37C while taking periodic optical density and fluorescence readings ([http://2012.igem.org/Team:Stanford-Brown/Protocols Protocols]). The results of these bulk assays are presented here.

The fluorescence reporter strain is mut3b GFP, which has a half-life of 33 hours. Since cell-cycle is on an order far less than 33 hours (~20 minutes), the fluorescence should not significantly decrease during the 1-2 hours of the microscope or bulk assays. Therefore, we expect a stepwise function in fluorescence readings (RFU). This matches the fluorescence reflected in the nrdP bulk assay, illustrated in Table 1.

When the fluorescence reading is divided against cell density, we expect a roughly sinusoidal graph: fluorescence increases as initiation of cell replication begins, then decreases as optical density increases at a greater rate than expression of fluorescence. The plotted ratio data follows such sinusoidal behavior; replication events are clearly visible right before t=20 and t=40, suggesting that promoter activity is cell-cycle dependent.

Table 1: Bulk assay results for nrdP

Usage and Biology

The nrd operon in E. coli expresses ribonucleotide reductase, an enzyme that reduces ribonucleotides into deoxyribonucleotides and is involved in bacterial cell cycle (Sun 1994). The promoter region is highly regulated and contains several DnaA boxes, indicating that activity is partly affected by initiation of DNA replication (Messer 2002). The promoter for this operon was found to begin activation during initiation of DNA replication and is cell-cycle dependent (Sun 1992).

This part is a cell-cycle dependent promoter tested in E. coli and can therefore be used in reporters for cellular growth or as a biosensor.

Sequence and Features