Cloning Of RecA Gene, KatB Gene And Their Biological Functions In λLysogenic Bacteria

The E.coli recA gene was amplified by PCR with a pair of primers. Then the PCR product was ligated with pUC18 and transformed into E.coli DH5a. A recombinant plasmid pUR4 carrying recA gene was constructed in the present study and then the plasmid was transformed into E.coli K12 (λ~+). Its biological functions in the lysogenic cells were determined with and without the UV induction. The results indicate that about 43% lysogenic cells with pUR4 would be induced to lyse.In order to know the mechanism that recA gene can induce the lysis of lysogenic cells, a ribosome binding sequence (rbs) GGAGAA was inserted into the upstream of recA gene. A new recombinant plasmid pUR6 carrying the recA gene was obtained. Then it was also transformed into E.coli K12 (λ~+) to study the biological function of recA gene. The results indicate that over 60% lysogenic cells with pUR6 could be induced to lyse under the same condition. The analysis with SDS-PAGE showed that the RecA protein was overexpressed in the E.coli K12 (λ~+)/pUR6.On the other hand, it was found that the X phage titers in lysogenic cultures with different plasmids varied a lot. The PFUs/mL of lysogenic cells with pUR4 and pUR6 were greatly more than that of the lysogenic cells with pUC18. It indicates that recA gene could induce the X prophage to enter lysis circle efficiently. Then the host cells were lysed and a great number of X phage particles were released At last. It also indicates that the RecA protein could interact with the λCI repressor without any activating signals, or by the activation of other signals, not only the ssDNA.A recombinant plasmid pUKB1 carrying the katB gene of Deinococcus radiodurans was constructed and transformed into lysogenic cell E.coli K12 (λ~+). It is found that Catalase B could inhibit the autogenesis of the lysogenic bacteria under non-induction condition in some measure. However, it could not inhibit the UV-induction of λ prophage at the same time.