#DnaA is an important protein for the initiation of DNA replication and also regulates gene transcription by binding to certain sequences in the DNA called DnaA boxes. In this project we examine the influence of DnaA boxes on the mioC promoter activity by constructing plasmids with changed sequences in the DnaA box and gfp as a reporter gene. The expression of mioC is detected by measurement of fluorescense. Plasmids containing the deactivated R8 DnaA box showed a better repression of the promoter compared to wild type cells. Transferring the same R8 mutation into other plasmids with simultaneous mutation of the ATP-DnaA box to the -35 consensus promoter sequence showed a derepression of the mioC promoter. In addition, the cooperative binding between R5 and R6 DnaA boxes in the mioC promoter was examined using a chromosomal mioC-lacZ fusion. This experiment showed that the DnaA N- terminus controlled by the lac promoter disturbed the cooperative binding to R5 and R6 resulting in derepression of the mioC promoter. The DnaA N-terminus also disturbed the initiation of replication of the oriC. This is indicated by the observation that, initially, the cell’s content of fully replicated genomes decreases as IPTG concentrations increase. Later, after the induction, high IPTG concentrations revealed an increasing level of genomes per cell and cell length also increased. This indicates that the N-terminus of DnaA protein may be inhibiting cell division.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) Bachelor el. kandidat|
|Udgivelsesdato||1 jan. 2007|
- DnaA, mioC promoter, gfp, B-galactosidase, DnaA boks, N-terminus, synkroni