The DnaA Protein Is Not The Only Limited Factor In The Initiation Of Chromosome Replication In Escherichia Coli

DNA replication in Escherichia coli usually starts at a unique site, the replication origin oriC, on the circular chromosome and proceeds bidirectionally to the terminus. The initiation event is tightly regulated, therefore the initiation of chromosome replication in Escherichia coli occurs once and only once in one generation. The dnaA gene with LacZ promoter was cloned into pUC19 using the Escherichia coli DH5a strain. The recombinant plasmid containing Lac-dnaA was named pUCA12. The Lac-dnaA from pUCA12 was inserted into the minichromosome pALO22, and we got the recombinant minichromosome named pDnaA. The minichromosome pALO22 and pDnaA were induced with IPTG, and Southern blotting and Western blotting showed that with the increasing of inducing time, the expression of DnaA protein was increased gradually. When the copy number of minichromosome pDnaA increased about four times of minichromosome pALO22, it reached the maximum. Therefore the result suggests that the DnaA protein is not the only limited factor in the initiation of chromosomereplication in Escherichia coli.