Plasmid RP4 :: Mucts 61 에 의한 E . coli K - 12 trpL ( Δatt ) trpE 유전자의 in vivo Cloning = In vivo Cloning of E . Coli K - 12 trpL ( Δatt ) trpE Gene Using the Plasmide RP4 Mucts 61

다국어 초록 (Multilingual Abstract)

In order to provide the trp gene source for the construction of recombinant multi-copy plasmid, and to study the expression of E. coli trp operon in the Aerobacter aerogenes 62-1, the regulation-free tryptophan operon of E. coli trpL (dart) trpEFBR, was cloned by in viva cloning technique using hybrid plasmid RP₄: : Mucts 61. When the chromosomal trpL (datt) trpE^(FBR) gene of E. coli LC110 was conjugally transferred by the plasmid RP₄: :Mucts 61 to E. coli LC501 and A. aerogenes 62-1, transfer frequencies were 3. 4×10^(-8) and 1. 2×10^(-9), respectively, Two E. coli transeonjugants, KUB101 and KUB102, and A. aerogeney transconjugant, AKB101, were isolated and characterized. In order to confirm the parental genetic character of the trp operon in the transconjugants, the activities of tryptophan synthetase and anthranilate synthetase were assayed. Transconjugants KUB102 and AKB101 had resistance to attenuator control and feedback inhibition by the tryptophan. However, KUB101 had resistance to feedback inhibition, but sensitive to attenuator control by the tryptophan, Drug resistance and Trp+ phenotype of the transconjugants were stable in the both complete and minimal medium containing ampicillin (100 ㎍). kanamycin (25 ㎍), and tetracycline (50 ㎍). The plasmid DNA of the predicted size (RP₄::Mucts 61-trp) was identified from the transconjugants by the agarose gel electrophoresis. On the basis of the results, it was concluded that the specific E. coli trp operon could be conveniently cloned in vivo in both E. coli and A. aerogenes 62-1 using hybrid plasmid RP₄: : Mucts 61 and that E. coli K-12 trp gene could be normally expressed in the A. aerogenes 62-1.