본 실험에서는 prokaryotic promoter중에서 강력하다고 알려진 phage $lambda$의 PL-promoter를 끼어 넣은 유전자 운반체를 개발하여 온도로써 이의 발현을 조절할 수 있는 대장균 속에 이주시켜 유전자 발현이 강력하면서도 온도로써 그 발현을 조절할 수 있는 host-vector system을 확립하였다. $lambda$ phage의 PL-promoter와 N gene이 함유된 BamHI--BglII fragment가 pBR322의 BamHI site에 삽입된 pPL81에서 N gene의 일부를 포함하는 부위인 899bp를 ClaI을 처리하여 제거하고 다시 연결시켜 pPL82를 개발하였으며, pPL82는 PL-promoter와 N gene의 일부를 가지므로 fused protein을 얻을 수 있다. 또한 pPL81을 HpaII로 처리하면 PL-promoter와 operator만을 갖는 273 bp가 나오며 이를 cohesive end가 같은 pBR322의 ClaI site에 삽입하였다. PL-promoter가 시계반대방향으로 들어간 것을 pPL83, 시계방향인 것을 pPL84이라 명명 하였으며 이들 plasmid는 EcoRI과 HindIII를 처리하면 PL-promoter만 있는 약 300 bp의 DNA조각을 얻을 수 있다. PL-promoter와 구조 유전자와의 거리에 따른 PL-promoter의 효율을 $eta$-lactamase 효소의 활성도로 조사한 결과 pPL81보다는 pPL82가 pPL82보다는 pPL83이 높았다.
In this study, series of expression vectors that contain PL-promoter of phage lambda were constructed. The PL-promoters in those plasmids were able to be controlled by temperature in the host Escherichia coli which contained temperature sensitive CI857 gene on the chromosome. Plasmid pPL81, which was constructed with the fragment containing the N gene and its PL-promoter of phage lambda into the BamHI site of pBR322, was digested with ClaI for the deletion of 899 bp and self-ligated to construct pPL82. The plasmid pPL82 is useful for constructing gene fusions whose product contained amino acids encoded by N gene. Besides the construction of pPL82, HpaII fragment containing only oLpL region of pPL81 was inserted into the ClaI site of pBR322. The plasmid with the PL promoter in the counterclockwise orientation was named as pPL83, and that in the clockwise orientation was named as pPL84. These plasmids carry portable promoter that can be separated by codigestion with EcoRI and HindIII restriction endonucleases. Since the, $eta$-lactamase genes of pPL81, pPL82 and pPL83 are located at the downstream of the PL-promoter, the $eta$-lactamase can be induced at $42^{circ}C$. The comparison of the production of $eta$-lactamase indicated that PL-promoter of pPL82 was more efficient than pPL81, and that of pPL83 was the most efficient. These results indicated that when the structural gene is closely located to the PL-promoter, the influence of PL-promoter is more effective.
