In order to develop useful plasmid vectors for Zymomonas cells, attempts were made to isolate some natural plasmids from Z.mobilis ATCC10988. Among a few plasmid isolated, a small plasmid having 2.7Kb in size was chosen and designated as pZM2.
A restriction map of pZM2 was made and found to contain one cleavage site for AvaI, BamHI, BglII and two for HindIII.
By introducing the replication origin of pZM2 into pBR325, hybrid plasmid vector was constructed and designated as pHZ22.
This vector contained chloramphenicol resistance gene as a genetic marker and proved to be stably maintained in Z.mobilis ZM4. Another trial was made to construct a better plasmid containing tetracycline resistance gene. This gene was isolated from RP4 and introduced into pHZ22 to make a new vector called pHZT224. Through a series of experiments, it was evident that these plasmid vector containing selectable markers of chloramphenicol and tetracycline resistance was a shuttle vector which was functional in Z.mobilis as well as in E.coli.
Z. mobilis ATCC10988 로부터 분리된 몇개의 plasmid 중 2.7Kb 의 적당한 크기를 갖는 pZM2 를 선정하여 Zymomonas 균주에 사용가능한 유전자 운반체의 개발을 시도하였다. 우선 pZM2 를 수종의 제한효소로 처리하여 절편의 크기로부터 유전자 지도를 작성하였다. pZM2 의 replicon 과 pBR325 의 chloramphenicol 저항표지를 포함한 잡종 plasmid인 pHZ22를 개발하고 이 vector가 숙주세포인 Z. mobilis ZM4 에서 replication 됨을 확인하였다.
또하나의 유전자표지로서 RP4 의 tetracycline저항표지를 분리하여 pHZ22 에 도입함으로써 pHZT224 를 제조하였는데 이vector는 Zymomonas로 전이되어 안정하게 유지되었다.
본 연구를 통하여 개발된 vector는 Z. mobilis 와 E. coli에 공히 작용하는 shuttle vector 로서 외부 유전자를 Zymomonas에 도입시킬 수 있는 유용한 유전자운반체임을 확인하였다.
