박테리아의 multi-copy plasmid vector에 사용될 trp gene source로서의 이용과 Aerobacter aerogenes에서의 E. coli trp operon 발현에 관한 정보를 연구할 목적으로 E. coli K-12의 $trpL({Delta}att)trpE^{FBR}$ 유전자를 hybrid plasmid $RP_4$::Mucts 61을 이용하여 E. coli LC501과 A. aerogenes 62-1에 각각 in vivo cloning 하였다. E. coli LC110의 염색체 $trpL({Delta}att)trpE^{FBR}$ 유전자를 hybrid plasmid $RP_4$::Mucts 61 에 의해 E. coli LC501과 A. aerogenes 62-1에 도입하였을 때의 transfer frequency는 각각 $3.4{ imes}10^{-8}$과 $1.2{ imes}10^{-9}$이었고, 이 때 E. coli transconjugant KUB101과 KUB102 그리 고 A. aerogenes 62-1의 transconjugant AKB101을 분리 하였다. 이들 transconjugant들에서 tryptophan synthetase와 anthranilate synthetase 활성을 측정하여 공여 E. coli trp operon의 유전적인 특성들이 그대로 유지되는지를 확인하였다. transconjugant KUB102와 AKB101은 공여주에서와 마찬가지로 트립토판에 의한 attenuator control과 feedback inhibition을 모두 받지 않았고, KUB101은 트립토판에 의 한 attenuator control은 받았으나 feedback inhibition은 받지 않았다. ampicillin($100;{mu}g/ml$), kanamycin($25;{mu}g/ml$) 그리고 tetracycline($50;{mu}g/ml$)이 각각 포함된 최소배지와 완전배지에서 transconjugant들의 항생제 저항성과 $Trp^+$ 형질은 안정하였으며 예상한 크기의 plasmid DNA ($RP_4$::Mucts 61-trp)가 transconjugant들에서 agarose 전기영동으로 확인되었다. 이상의 결과로부터 E. coli trp operon을 간편하게 E. coli와 A. aerogenes에서 in vivo cloning할 수 있고 E. coli trp operon이 A. aerogenes에서 정상적으로 발현될 수 있음을 확인하였다.
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 (${Delta}att$) $trpE^{FBR}$, was cloned by in vivo cloning technique using hybrid plasmid $RP_4$::Mucts 61. When the chromosomal trpL (${Delta}att$) $trpE^{FBR}$ gene of E. coli LC110 was conjugally transferred by the plasmid $RP_4$::Mucts 61 to E. coli LC501 and A. aerogenes 62-1, transfer frequencies were $3.4{ imes}10^{-8}$ and $1.2{ imes}10^{-9}$, respectively. Two E. coli transconjugants, KUB101 and KUB102, and A. aerogenes 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 ($100;{mu}g$), kanamycin ($25;{mu}g$), and tetracycline ($50;{mu}g$). The plasmid DNA of the predicted size ($RP_4$::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 $RP_4$::Mucts 61 and that E. coli K-12 trp gene could be normally expressed in the A. aerogenes 62-1.
